Immune responses in thymus derived (T) cell-depleted mouse spleen cultures were stimulated by the addition of either syngeneic T cells immunized with antigen, or allogeneic T cells reactive against precursor antibody-forming cells. Both require the presence of specific antigen in cultures. At low T cell concentrations, (< 106), allogeneic T cells are more effective in stimulating immune responses than the syngeneic T cells, however, at higher cell numbers (> 106), allogeneic T cells suppress the response. The stimulation of in vitro immune responses to heterologous erythrocyte antigens is mediated by nonspecific activity released from antigen-stimulated T cells in culture supernatants. Fractionation of culture supernatants prepared in serum-free media, by Sephadex G-100 chromatography, revealed the presence of three thymus cell-replacing factors (TRF). The fractionation profiles of supernatant activities prepared from syngeneic and allogeneic T cells were identical. TRF-I from both T cell sources has a molecular weight around 150,000, is labile to heat, periodate, and trypsin, and requires the presence of θ-positive cells for production in culture. TRF-I does not appear to be immunoglobulin in nature. TRF-II (m.w. 15,000 to 40,000), and TRF-III (m.w. < 7000) do not require the presence of θ-positive cells for production in culture. Syngeneic and allogeneic T cells also released a factor (m.w. 10,000 to 20,000) that suppressed the induction of normal in vitro immune responses.