The three mitogenic anti-T3 antibodies, UCHT1, anti-Leu-4 and WT-32, all produce a rapid increase in T cell intracellular Ca2+ ( [Ca2+]i) in all individuals, as measured by quin 2 tetra-acetoxymethyl ester fluorescence. This indicates that the lack of responsiveness of approximately 30% of individuals to UCHT1 in proliferation assays is not due to failure of the antibody to elicit Ca2+ mobilization and that a rise in [Ca2+]i is per se not adequate to induce cell division. Another mitogenic antibody, WT-31, which is directed against the constant portion of the T cell receptor, did not, however, produce a rapid calcium rise in peripheral blood T cells. The clone HA1.7 gave a similar Ca2+ response to UCHT1. WT-31 did not induce a rise in [Ca2+]i, nor did the specific antigen to which the clone responded. Accessory cells may be required to induce Ca2+ mobilization with these ligands. There was no response to IL2, or an antibody (anti-Tac) to the IL2 receptor. In contrast to peripheral blood T cells treatment of HA1.7 with WT-31 led to an enhancement of the calcium response to subsequent UCHT1 addition. Furthermore, cross-linking of WT-31 on the surface of HA1.7 cells did produce a small rise in [Ca2+]i. The IL2-independent malignant T cell line, HUT78, exhibited a calcium response to both UCHT1 and WT-32. Both of these responses occurred without cross-linking. The T cell receptor is closely associated with cell-surface proteins, including the T3 antigen and these studies confirm the importance of the T3 antigen in T cell activation. They also suggest that the relationship between the T cell receptor and the T3 antigen may vary in T cells in different proliferative states.