Cock spermatozoa were incubated for 6 and 24 hr at 41 C with minimum essential medium (MEM), tissue culture fluid (TCF) removed from tissue cultured cells and in the presence of tissue cultured cells (TCC). the TCC were obtained from whole 9-day-old chick embryos. Fertility following intravaginal artificial insemination was 75, 77, and 80% for semen incubated at 41 C for 6 hr in MEM, TCF, and TCC, respectively. Fertility of semen incubated for 24 hr was approximately 50% for all three treatment groups. The MEM alone was as beneficial as TCF or TCC in maintaining in vitro sperm survival. These observations provide a method for maintaining in vitro the fertilizing capacity (as evaluated by intravaginal insemination) of large numbers of cock spermatozoa under conditions of temperature comparable to those of the hen's oviduct.