The experiments reported herein have characterized the signaling pathway leading to stimulation of type I protein kinase A isozyme (PKA-I) activity during the early events of Ag receptor-mediated T cell activation. Inhibitor studies demonstrated that receptor-initiated activation of nonreceptor protein tyrosine kinases, phosphorylation and activation of phospholipase C-gamma 1, and activation of protein kinase C occur temporally and precede PKA-I activation. Bypass of both the TCR/CD3 complex and IL-1R and direct activation of protein kinase C by a phorbol ester can also activate PKA-I. To confirm that PKA-I activation via the TCR/CD3 complex and IL-1R requires antecedent protein tyrosine kinase-catalyzed phosphorylation of phospholipase C-gamma 1, we used wild-type and CD45-deficient (mutant J45.01) Jurkat T cell lines. Unlike wild-type Jurkat T cells, the absence of CD45 tyrosine phosphatase resulted in the failure of receptor-mediated activation of PKA-I activity and of IL-2 mRNA transcription in the mutant J45.01 Jurkat cell line. In conclusion, our data support the concept that a signal derived from ligand binding to both the TCR/CD3 complex and IL-1R receptor mediates rapid activation of the PKA-I isozyme in primary T lymphocytes by sequential activation of an intracellular pathway comprised of CD45 phosphatase/protein tyrosine kinase/polyphosphoinositide/Ca2+/protein kinase C pathway rather than via the conventional surface receptor/stimulatory G protein system.