An established Glossina cell line with a modified insect tissue culture medium supports transformation of bloodstream forms and continuous growth of procyclic forms of Trypanosoma congolense. Midmastigotes differentiate reproducibly to epimastigotes, but metacyclic forms are not produced, as shown by the absence of infectivity to mice and rats. Cultivation of T. congolense may require vector-specific factors, since Drosophila cells do not support growth. The respiration/inhibition pattern of T. congolense bloodstream and procyclic forms closely resembles that of T. brucei, except for the absence of an initial cyanide-insensitive "phase" in established culture forms. The cattle pathogen, Trypanosoma congolense, can readily be grown in blood-agar medium (Tobie, 1958; Yesufu, 1970) and in primary organ cultures derived from various tissues of the vector, Glossina (Trager, 1959; Trager and Krassner, 1967; Cunningham, 1973). Infectivity for mammalian hosts is lost after a short time in vitro (Tobie, 1958), as the bloodstream parasites transform to procyclic culture trypomastigotes (Hoare and Wallace, 1966) or midmastigotes (Trager, 1974), which correspond to midgut forms of the tsetse fly. Further development to the epimastigote and infective, metacyclic (metatrypomastigote) stage has only rarely been observed. On the other hand, Trypanosoma vivax grown in tsetse fly organ cultures has been observed to complete the entire developmental cycle with the production of metacyclic forms (Trager, 1959). Since defined and semi-defined media do not support growth of T. congolense (Steiger, unpublished), experiments described here have been carried out to see whether this parasite can be maintained in the presence of a Glossina cell strain. In its reproducibility and simplicity such a system would be superior to organ cultures. Preliminary data of our study were preReceived for publication 1 February 1977. * Recipient of a Fellowship from the "Swiss National Foundation," Nr. 831.372.75; with additional support from the "Basle Foundation for Experimental Zoology." Present address: ICP, Universite Catholique de Louvain, 75, Ave. Hippocrate, B-1200 Bruxelles. t Laboratory of Parasitology, The Rockefeller University, New York, New York 10021. t From the Department of Entomology, Walter Reed Army Institute of Research, Washington, D.C. 20012. sented at the 25th Annual Meeting of the American Society of Tropical Medicine and Hygiene (Philadelphia, 1976). MATERIALS AND METHODS Bloodforms of Trypanosoma congolense (strain TREU 1183) were obtained from Dr. B. M. Honigberg. This strain had been isolated in 1970 from Glossina pallidipes in Lugala, Uganda. NCS mice (20 to 25 g) from the Rockefeller University colony and Sprague-Dawley rats (150 g) were infected i.p. with thawed stabilates prepared from this strain after a total of 8 to 10 mouse passages. Blood-agar slants were seeded with 0.1 ml of parasitized whole blood aseptically removed by heart puncture on days 8 to 10 after inoculation (108 parasites/ml of blood). These were prepared according to Tobie (1958), except for using defibrinated bovine blood (GIBCO) and different liquid overlays: HEPES buffered TC-199, Grace's insect T.C. medium (GM), Hanks' BSS (all from GIBCO). The cultures were kept at 27 C and transferred weekly. A cell line derived from larval tissues of Glossina morsitans (Schneider, unpublished) was grown in a modification of Schneider's Drosophila medium (SM) (Table I) in Falcon tissue flasks (# 3013) at 25 C. Just before the monolayer was confluent (days 8-12), the cells were gently pipetted off, suspended in fresh SM in 1:6 or 1:8 dilutions and transferred to new flasks. Trypanosoma congolense was grown in flasks with confluent monolayers (days 14-20; ~ 2.5 X 104 cells/cm2). Initial cultures of T. congolense were started with a drop of parasitized blood or with midmastigotes harvested after 8 to 12 passages from Tobie's medium with a GM overlay. Weekly subpassages were made as follows: after removal of the supernatant fluid (conditioned medium) 3.5 ml of fresh SM and 0.5 ml of an inoculum containing cultured parasites adjusted to a final cell concentration of 2-5 X 105/ml were added. Identical aliquots of parasites were grown in conditioned medium cleared by centrifugation and filter-sterilized through Millipore Millex disposable filter units (0.22 am), and in a Drosophila cell line (Nr. 3;