Conditioned culture media of ovine placental tissues or fetal skeletal muscle collected at Days 60, 100 and 140 of pregnancy were tested for their ability to inhibit in vitro lymphocyte proliferation. All conditioned cultures contained low-molecular-weight factors which suppressed [3H]Tdr uptake by PHA-stimulated sheep lymphocytes. Non-dialyzable, inhibitory molecules were released only by placental tissues, with the greatest activity occurring for fetal cotyledonary placenta at Days 100 and 140 of gestation. Dialyzed conditioned culture medium of Day 100 cotyledonary placenta suppressed lymphocyte proliferation stimulated by PHA, PWM and MLR, but had no effect on ConA-stimulated cultures. Inhibitory actions were effective only during the first 24 h of lymphocyte activation and were abrogated with exogenous IL-2. Size-fractionation by gel filtration chromatography of the conditioned medium resolved the inhibitory activity into a peak of molecular weight greater than 4 X 10(6) Da and another peak with estimated molecular weight range of 46-162 kDa. Inhibitory action of the high molecular weight fraction was destroyed by sodium periodate oxidation whereas the lower molecular weight peak was sensitive to pronase. Presence of these immunosuppressive factors at the maternal-conceptus interface may contribute to down-regulation of local immune functions in the uterus and survival of the allogeneic conceptus.