Specific activation of CD4 T cells involves recognition of a peptide: MHC class II complex by the heterodimeric alpha beta TCR and its CD4 co-receptor. The activation of T cells initiates a signal transduction cascade that includes a substantial and rapid increase in cytosolic calcium. In this work we study the role of the interactions between the TCR and the CD4 molecule in inducing this [Ca2+]i increase in the cloned CD4 T-cell line D10. Ligating CD3 or the TCR with mAb leads to proliferation of this cloned line and an increase in intracellular free calcium. An exceptional antibody, 16A, was able to stimulate proliferation but did not induce an increase in intracellular free calcium, even when extensively cross-linked with anti-Ig. However, when 16A was cross-linked to CD4 a rise in [Ca2+]i was observed. This demonstrated the ability of TCR-CD4 interactions to trigger a [Ca2+]i response in a situation where no signal was obtained from TCR-TCR interactions. Because CD4 molecules can associate with the TCR, we also studied whether CD4 is involved in the observed increases in intracellular free calcium obtained with other anti-TCR antibodies. This was examined in CD4-negative T cells transfected with cDNA encoding the D10 TCR. These cells could be induced to flux calcium by the same anti-TCR antibodies that gave this response in D10 cells, and again 16A failed to induce such a response. In the transfectants, anti-CD3 antibodies, in contrast to TCR antibodies, could induce a calcium signal in the absence of cross-linking by anti-Ig indicating that CD3 antibodies may signal distinctly from alpha beta TCR ligation. These studies document that antibodies binding different TCR/CD3 epitopes signal distinctively, and that CD4 can participate in, but is not absolutely required for, the induction of increased intracellular calcium.