Protein tyrosine kinases play fundamental roles in the transduction of signals that regulate cell growth, differentiation, and functional responses to a diversity of external stimuli. It is therefore likely that understanding protein tyrosine kinase activity in NK cells will be crucial in further defining the intracellular regulation of their unique and specialized functions. We investigated the role of protein tyrosine phosphorylation in receptor-mediated signal transduction using stimuli known to play major roles in regulating NK cell activation. Immunoblot analyses with antiphosphotyrosine antibodies demonstrated that IL-2, a potent stimulus for NK cell proliferation and an agent that enhances NK cytotoxic function, induced the tyrosine phosphorylation of at least eight proteins in clonal CD16+/CD3-human NK cells. In contrast, IL-4, which modulates NK cell function without inducing proliferation, had no apparent effect on protein tyrosine phosphorylation. Because protein kinase C (PKC) activation plays a prominent, yet distinct role in NK cell-mediated cytolytic reactions, we next investigated whether PKC activation affects NK cell protein tyrosine phosphorylation. Surprisingly, PKC-activating agents, including the phorbol esters 12-O-tetradecanoylphorbol-13-acetate and 4 beta-phorbol 12, 13-didecanoate, as well as the synthetic diacylglycerol,1-oleoyl-2-acetylglycerol, also induced the tyrosine phosphorylation of a distinct set of proteins. The 4 beta-phorbol 12,13-didecanoate homolog, 4 alpha-phorbol 12,13-didecanoate, which does not activate PKC, also failed to induce protein tyrosine phosphorylation. Further, the PKC inhibitor, 1-O-hexadecyl-2-O-methylglycerol blocked tyrosine phosphorylation induced by 1-oleoyl-2-acetylglycerol. In subsequent studies, both CD8+ and CD8- NK clones were found to express the src-family tyrosine kinase, p56lck, which was detected by immunoblot analysis with anti-p56lck antiserum. In both types of clonal NK cell lines, IL-2 and 12-O-tetradecanoyl-phorbol appeared to stimulate the differential phosphorylation of p56lck as evidenced by the appearance of higher molecular mass isoforms on SDS-polyacrylamide gels. Thus, our results identify and characterize a potential role for tyrosine phosphorylation and for the lymphocyte-specific tyrosine kinase p56lck in the signaling events that regulate NK cell activation.