The murine T-lymphoma cell line LBRM-33 is known to require synergistic signals delivered through the antigen receptor (T1-CD3) complex, together with interleukin 1 (IL-1), for activation of IL-2 gene expression and IL-2 production. Although 12-0-tetradecanoylphorbol-13-acetate (TPA) was capable of replacing IL-1 as an activating stimulus under certain conditions, biologic studies indicated that TPA failed to synergize with Ti-CD3-dependent stimuli under conditions in which IL-1 was clearly active. Acute exposure to TPA and other active phorbol esters resulted in a concentration-dependent inhibition of the increases in phosphoinositide hydrolysis and intracellular free Ca2+ concentration stimulated by phytohemagglutinin or anti-Ti antibodies. TPA treatment induced no direct alteration of phospholipase C enzymatic activities in LBRM-33 cells. In contrast, both T1-CD3 cross-linkage and TPA rapidly stimulated the phosphorylation of identical CD3 complex polypeptides, presumably via activation of protein kinase C. Exposure of LBRM-33 cells to TPA resulted in a time-dependent, partial down-regulation of surface T,-CD3 expression. Thus, TPA treatment inhibited the responsiveness of LBRM-33 cells to T1-CD3-dependent stimuli by inducing an early desensitization of Ti-CD3 receptors, followed by a decrease in membrane receptor expression. These studies indicate that phorbol esters deliver bidirectional signals that both inhibit T1-CD3-dependent phosphoinositide hydrolysis and augment IL-2 production in LBRM-33 cells.The T-cell antigen receptor is a multimeric structure consisting of a clonotypic, disulfide-linked heterodimer (Ti) noncovalently associated with a series of invariant transmembrane proteins collectively termed CD3 (40,41