MHC class II-positive T cells are found in tissues involved in autoimmune and infectious disorders. Because stimulation of class II molecules by mAb or bacterial superantigens induces protein tyrosine phosphorylation through activation of PTK3 in T cells, we hypothesized that class II signals play a regulatory function in T cell activation. Here, we show that cross-linking HLA-DR and -DP but not -DQ molecules by immobilized mAb enhanced proliferative T cell responses to IL-2. In contrast, class II stimulation had no effect on IL-4-induced proliferation. The costimulatory effect was most pronounced at low concentrations of IL-2, was blocked by IL-2R mAb, and was at least partly mediated through an up-regulation of IL-2 high affinity receptors. As expected, activation of IL-2R by IL-2 induced tyrosine phosphorylation of several proteins including p56lck, and class II cross-linking by mAb induced tyrosine phosphorylation of specific substrates including PLC-gamma 1. Combined stimulation of IL-2R and class II molecules had an additive effect on tyrosine phosphorylation. Pretreatment of T cells with a protein tyrosine kinase inhibitor, herbimycin A, inhibited IL-2 and class II-induced proliferation suggesting that class II costimulation of IL-2 responses may involve activation of tyrosine kinases. Taken together, the present data suggest that extensive cross-linking of class II molecules delivers costimulatory signals that enhance IL-2 sensitivity in human T cells.