To understand the proliferation/differentiation of immature thymocytes which have not express T cell antigen receptor (TCR), we studied whether Thy-1 has signal-transducing capacity. Thy-1+ CD3-TCR- cells including thymocytes from BALB/c embryos and SCID mice and nude mouse splenic cells did not show proliferative responses in the culture with anti-Thy-1 (G7) plus phorbol myristate acetate (PMA), whereas Thy-1+ CD3+ cells from normal thymus or spleen did show a response to them. Since Thy-1-mediated activation is suggested to require co-expression of the CD3-TCR complex, we compared the T cell proliferative response in mature T cells stimulated with anti-Thy-1 (G7) and anti-CD3-epsilon (2C11). Under the presence of PMA or IL-2, accessory cell-depleted splenic T cells were cultured with G7 or 2C11. PMA augmented the proliferative response of splenic T cells cultured with G7 much more than that with 2C11. IL-2, however, showed reciprocal effect on the proliferation of G7 and 2C11-treated splenic T cells. These data suggest that signals triggered via Thy-1 and CD3-epsilon may provide a distinct intracellular pathway for T cell activation.