Five strains of Leishmania, two of L. donovani, and three strains from human cutaneous lesions (L. mexicana, L. braziliensis, and L. braziliensis pifanoi), have been maintained for at least a year with apparent multiplication in the intracytoplasmic leishmanial stage in a dog sarcoma cell line. When temperature of incubation was 36 C, L. donovani were leishmaniform and flagellates were absent. At 33 C, extraand intracellular parasites were elongate and flagellates were numerous. With the "cutaneous" strains leishmaniforms were present at 33 C and flagellates appeared at 28 C. At 36 C, parasites of the two strains of L. braziliensis disappeared but those of L. mexicana survived. Leishmaniform infections of cultured cells with "cutaneous" strains were further characterized by large numbers of parasites within cytoplasmic vacuoles, while infections with L. donovani were characterized by relatively few parasites apparently embedded in the cytoplasm. Many types of cell culture have been infected with Leishmania spp., although continuous cultivation of intracellular leishmanial forms in tissue cultures has been an elusive goal. In this laboratory, for example, human amniotic cells were readily infected, but the intracellular leishmaniforms eventually disappeared from the cultures (Frothingham and Lehtimaki, 1967). However, continuous intracellular growth in vitro in the leishmanial form was described by Lamy and co-workers (1964), who studied L. donovani in a cell line derived from a dog's sarcoma. We report here on the use of this cell line for prolonged culture of intracellular leishmaniforms of two strains of L. donovani and of three strains of Leishmania derived from patients with cutaneous disease. MATERIALS AND METHODS Details of most of the methods and of the origins of some of the parasites have been described (Frothingham and Lehtimaki, 1967). L. donovani: Strain P, isolated by Dr. S. C. Pan from a Pakistani with visceral infection, was maintained in NNN cultures for more than a decade; cell culture passages were initiated with leptomonads. Strain S, from a Sudanese with visceral infection, was isolated by Dr. Harry Browne and maintained in hamsters until infection of cell cultures was initiated with infected ground splenic tissue. Strain G, from a dog with visceral disease, probably acquired in Greece (Theran and Ling, 1967), was isolated by us and maintained in hamReceived for publication 7 June 1968. * Supported in part by Research Grant AI-01023 and Training Grant AI-177 from the NIAID, U. S. Public Health Service. sters until infection of cell cultures was initiated with ground spleen. Other Leishmania spp.: Three strains originated from patients with cutaneous disease. L. mexicana, a strain described by Garnham and Lewis (1959), was furnished by Dr. Marshall Hertig who had maintained it for several years in hamsters; infection of cell cultures was started with flagellated forms from our 26th NNN passage. L. braziliensis pifanoi, from a patient with difusa ype of cutaneous leishmaniasis, was furnished by Dr. J. Convit. Inoculum for the present experiments with this strain was derived from nutrient fluid and cells scraped from infected cultures of human amniotic cells (Frothingham and Lehtimaki, 1967). L. braziliensis (sensu lato) was isolated by Dr. Bryce Walton in 1965 from a cutaneous lesion on a North American infected in Panama. Infection of cell cultures with this strain was initiated with flagellated forms from the 19th passage in NNN. The dog sarcoma cell line, kindly supplied by Dr. Lamy, was maintained at 36 C by conventional methods in stationary bottles. Cultures were nourished with Parker's medium 199 containing 10% fetal bovine serum (FBS) previously heated at 56 C for 30 min. For experiments, cells were grown on coverslips in Leighton tubes in nutrient medium containing 5 to 20% fetal bovine serum. Nutrient medium was changed twice a week. Incubation was at 28, 33, or 36 C ? 1 C. Cultures were examined microscopically, and cover slips were studied after fixation with Zenker's acetic acid and staining in Giemsa's. The percentage of cells infected was determined as already described (Frothingham and Lehtimaki, 1967). The average number of organisms per infected cell was determined by counting the number of parasites in 100 consecutively encountered infected cells. The number of cells per culture was calculated from the number of cells in an apparently representative area of the cell sheet approximating 1/450th of the total area. The number of intracellular organisms per culture was calculated from all three foregoing values.