Studies on the effect of rat testicular interstitial fluid (IF) on T-cell function have reported both stimulatory and inhibitory actions. Specific cytokines produced within the testis, particularly interleukin-1 (IL-1) and transforming growth factor beta (TGFbeta), may contribute to these apparently conflicting observations. In proliferation assays employing lectin- or antibody-activated thymocytes or mature T cells in vitro, adult rat testicular IF stimulated T-cell activation and/or proliferation at low assay doses and was inhibitory at higher doses. The stimulatory activity was blocked by recombinant IL-1 receptor antagonist. The inhibitory activity was not affected by a polyspecific TGFbeta antiserum. The biological characteristics of the inhibitor were distinct from those of a similar, but considerably less potent, activity in platelet-depleted serum. These data demonstrate that rat testicular IF contains biologically significant concentrations of IL-1 but has a predominantly inhibitory action on T-cell responses. The factor predominantly responsible for this inhibitory activity displays a relatively large apparent molecular weight, is protease sensitive and partially heat labile, but does not appear to be one of the known mammalian TGFbeta isoforms.