High molecular (designated Sv,>3K) and low molecular (designated Sv, c3K) fractions were prepared by filtration of porcine vitreous extracts through a membrane filter with a molecular weight cut-off of 3,000 daltons. Both fractions inhibited mitogen-stimulated proliferation of lymphocytes from rat spleen, as determined by [3H]-thymidine incorporation. The inhibitory activity of Sv,>3K was markedly enhanced by acid treatment and abolished almost completely by incubation with anti-tiansfonning growth factor beta (TGF-β) antibody. The major immunosuppressive factor in Sv,>3K was therefore concluded to be TGF-βOn the other hand, the inhibitory activity of Sv,<3K was not affected by acid treatment or by incubation with anti-TGF-p antibody but was decreased by proteolytic treatment. From these results the immunosuppressive factor in Sv,3K and Sv,<3K were reversible and therefore not cytotoxic. Both factors seemed to suppress lymphoproliferation without affecting the expression of Interleukin-2 receptor, CD4 and CD8 on the cell surface of cultured lymphocytes.