To examine which cell surface molecules can operate as transducers of activation signals to early fetal thymocytes, we analyzed the ability of mAb to CD3 and Thy-1 to induce fetal thymocyte activation. Both proliferation and lymphokine secretion were used as measures of activation. We show that anti-CD3 antibodies induce activation of fetal thymocytes as early as day 13 of fetal thymus development, 2 days before CD3 can be detected by flow cytometry. In addition, an alternative activation signal can be delivered to fetal thymocytes through the Thy-1 molecule as early as it is expressed, i.e., day 13. Both CD3- and Thy-1-mediated activation of day 15 fetal thymocytes results in expansion of cells expressing a CD3-gamma delta receptor complex; no CD3-alpha beta receptor complex could be detected. IL-2 production induced by CD3- and Thy-1-induced activation of fetal thymocytes is evident at the 13th day of gestation. Finally, an additional lymphokine B cell stimulatory factor-1 (BSF-1)/IL-4 (so far known only to be produced by mature CD3- cells), is also produced by fetal thymocytes. The results demonstrate that at least two cell surface molecules, Thy-1 and CD3, can function as pathways of activation in fetal thymocytes, and that at least two lymphokines, IL-2 and BSF-1/IL-4, are produced upon activation. These findings may well reflect a role for the early appearance of CD3- cells in thymus ontogeny.