Although significant progress has been made in identifying the immunosuppressive factors in seminal plasma (SP) and their possible action in vitro, the potential role of SP in naturally occurring normal immune responses in vivo is less certain. Human SP or its fractions, administered 5 or 10 times at 3-day intervals into the mouse vagina simultaneously with washed human sperm or sheep red blood cells, suppressed the delayed-type hypersensitivity reactions to footpad injection of the antigens. However, SP failed to suppress antibody formation to these antigens in the applied experimental conditions. In an in vitro study, SP suppressed the proliferation of human peripheral blood mononuclear cells to phytohemagglutinin. This suppression of in vitro proliferation by SP was mediated by materials of both low (LMW) and high molecular weight (HMW). Among the HMW materials, a factor with approximately 1,500 kilodaltons, partially purified by DEAE-cellulose chromatography and gel filtration, exhibited the most potent suppressor activity in vitro and in vivo. The suppressive activities of SP or its fractions were not abolished by treatment with both heat (95 °C for 10 min) and trypsin (1.0 mg/ml, 37 °C for 4 h). These findings indicate that SP could contribute to the development of suppressed cellular immunity to sperm; immunosuppressive actions of SP are mediated by at least two distinct factors in vivo as well as in vitro.