With dimethyl sulfoxide as the cryoprotectant, axenic cultures of three isolates (JBIV, JBV, JBVI) of the Jones' Barn (JB) Trichomonas gallinae strain retained their original virulence for pigeons after 5, 5.5, and 7 years of storage at subzero temperatures, primarily in liquid nitrogen at -196 C. The closely comparable times required by noncloned and cloned populations of the JBVI trichomonads to kill Trichomonas-free, nonimmune pigeons indicated the homogeneity of the JB strain with respect to virulence. Although higher pathogenicity for birds was retained by the JBVI parasites maintained for 1 year by serial intraperitoneal passages in mice than by those kept for the same length of time in nonliving media, it was much lower than the virulence typical of the JB strain. The flagellates transferred in mice did not kill the avian hosts and their original pathogenicity levels could not be restored by several bird-to-bird passages. Nearly full virulence for pigeons was retained by the JBVI isolate which had been maintained for up to 1 year in the presence of chick liver cell cultures. Cell culture medium-CPLM mixture had no virulence-preserving effect. JBV trichomonads kept for 22.5 weeks in a CPLM-type medium, which contained no added carbohydrates and in which little division of the parasites took place, appeared to retain their pathogenicity for birds for a longer time than when maintained in the conventional CPLM and fluid thioglycollate media. The first report on the effects of various laboratory procedures on the virulence for pigeons of the Jones' Barn (JB) strain of Trichomonas gallinae (Rivolta) summarized the experimental results obtained during the early stages of our study (Stabler et al., 1964). According to these results: (1) prolonged (19 to 21 weeks) axenic cultivation of the JB parasites by serial transfers in nonliving media at 37 + 0.5 C caused pathogenicity loss; (2) trichReceived for publication 12 March 1970. * This investigation was supported by Research Grant AI00742, from the NIAID, U. S. Public Health Service. t Department of Biology, Colorado College, Colorado Springs, Colorado 80903. t Permanent address: Department of Protozoology, Institute of Parasitology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia. Dr. Kulda spent 2 years as Research Associate in the Department of Zoology, University of Massachusetts at Amherst. During this period he was supported mainly by Research Grants AI00742-13, 14, from NIAID; part of his support was provided by Biomedical Sciences Support Grant 5-SO5-FR07083-03, U. S. Public Health Service. ? The help of Mrs. Mary Tarr Hadley in certain parts of this investigation is hereby acknowledged. omonads stored at -20 or -72 C for 52 weeks retained their original virulence level; (3) pathogenicity loss was enhanced by the inclusion of penicillin and streptomycin in the original isolation medium (the JB flagellates ceased to kill nonimmune pigeons after 9 weeks of cultivation at 37 ? 0.3 C). These findings, which served as the basis for subsequent immunologic (Goldman and Honigberg, 1968; Honigberg and Goldman, 1968; Stepkowski and Honigberg, in Honigberg, 1969) and biochemical (Honigberg and Livingston, 1968) studies, suggested undertaking additional investigations of the ways whereby different laboratory procedures might affect the virulence of the JB strain. The present paper deals with the results obtained in the course of these investiga-