Activation of protein kinase C has been shown to be involved in the activation pathway of many cell types. Recently, a number of investigations have suggested that protein kinase C plays an essential role in T lymphocyte activation. The recent synthesis of the protein kinase inhibitors, H-7 and HA1004, have now made possible a new approach for testing the relevance of protein kinase C in T cell activation and proliferation. We now report that the antigen-induced and interleukin-2-induced proliferation of murine T cell lines can be consistently inhibited by the protein kinase C inhibitor, H-7. HA1004, a somewhat more potent inhibitor of cyclic nucleotide-dependent protein kinases, but a significantly weaker inhibitor of protein kinase C than H-7, demonstrated no consistent inhibition of these T cell responses. These results represent a further demonstration that protein kinase C plays an essential role in the activation of T cells.