Follicular fluid from bovine follicles at various stages of the estrous cycle and cysts were tested in vitro for immunosuppressive activity. Mitogen-stimulated lymphocyte assays and the mixed lymphocyte reaction were used as test procedures and thymidine uptake was monitored. Follicular fluid and cyst fluid inhibited the deoxyribonucleic acid synthesis response of bovine lymphocytes to mitogenic and antigenic stimulation. The nature of the suppression varied with the mitogen or antigen assay used, with the estrual stage, with size of follicle within the diestrual stage, and with the type of ovarian cyst. Blood sera from estrual or diestrual cows were not immunosuppressive. Heating (56 degrees C for 30 min) follicular fluid from normal follicles and cysts destroyed most of its suppressive activity toward Concanavalin A-stimulated lymphocytes but did not suppress that in luteal cyst fluid. The suppressive activity in follicular fluid toward phytohemagglutinin-stimulated lymphocytes was more stable to heat than that against Concanavalin A-stimulated lymphocytes. Because the immunosuppressive activity in follicular fluid was heat labile, estrogen, progesterone, and other steroids were not the active suppressive molecules. Proposed roles for these immunosuppressive substances are hypothesized in preventing autoantibody formation to the zona pellucida and in the inflammatory reaction leading to ovulation.