We have previously demonstrated that both human CD4+ and CD8+ T lymphocytes produce enhanced levels of luteinizing hormone-releasing hormone (LHRH) mRNA and peptide upon stimulation with monoclonal antibody directed at the CD3 component of the T cell receptor for antigen (TCR) or mitogenic lectin. In the current study, we define the signaling pathways that control TCR-mediated LHRH production by using agents known to affect distinct signals, and compare the messenger systems required for LHRH response to other T-cell-associated activation responses, such as expression of CD69 and interleukin-2 receptor (IL-2R) molecules and production of interleukin-2 (IL-2). Results indicate that the activation of protein kinase C (PKC) is essential for LHRH production by previously nonstimulated T cells, not increased concentration of cytosolic-free calcium ([Ca2+]i). Phorbol ester (PMA), a direct activator of PKC, provoked LHRH production and cell surface expression of CD69 and IL-2R molecules by T cells, but not IL-2 synthesis. The synthesis of IL-2 by T cells required costimulation with PMA and ionomycin, a Ca2+ ionophore. Consistent with these observations, H7, an inhibitor of PKC, prevented T cells from producing LHRH upon activation with mitogen. However, LHRH production was not suppressed by HA1004, which inhibits all cyclic nucleotide-dependent protein kinases except for PKC. Genistein, a selective inhibitor of protein tyrosine kinase, blocked PMA-induced increase in LHRH production, but not CD69 and IL-2R expression, suggesting that protein tyrosine phosphorylation events distal from PKC activation may play a role in regulating LHRH response.