Several recent observations suggest that successful rearrangement of the T cell receptor (TCR) beta locus induces several important events in thymocyte maturation. Allelic exclusion is achieved by interruption of further rearrangement of the beta locus, and CD4-8- interleukin (IL)-2R+ cells enter the CD4+8+IL-2R- stage. The actual molecular events regulating this important control point are unknown, but may be related to the expression of the TCR-beta locus in immature CD4-8- thymocytes. It is not clear whether maturation is induced by intracellular appearance of TCR-beta chain or by signal transduction through an immature TCR complex on the thymocyte membrane, possibly involving TCR-beta chain homodimers and CD3. Here we show that early addition of anti-CD3 mAb to fetal thymic organ cultures induces all known events associated with the acquisition of the CD4+8+ stage. Expression of CD4 and CD8 is accelerated, IL-2R alpha is downregulated, and the cells fail to produce TCR-beta, possibly based on premature cessation of beta gene rearrangement. Upon stimulation with anti-CD3 antibodies, we see calcium mobilization in 15% of all CD4-8- thymocytes with no detectable surface TCR expression. These results suggest that functional CD3 is expressed on immature thymocytes at very low concentrations before the appearance of a complete TCR-beta chain. Ligation of CD3 at this stage may mimic the maturation signal normally generated by the immature TCR-beta homodimer-CD3 complex. The results are consistent with the notion that acquisition of the CD4+8+ stage involves signal transduction through an immature TCR complex. Later in thymocyte development, ligation of CD3 results in deletion of CD4+8+ cells. Thus, signal transduction through CD3 may result in entirely different cellular responses, depending on the stage of thymocyte differentiation. These results suggest an involvement of CD3 as a link in signal transduction for at least two different decision points in the development of a thymocyte.