H uman T lymphocytes possess both the type I and II isozymes of protein kinase A (PKA). The type I (PKA-I) isozyme is predominantly associated with the plasma membrane, whereas the type II (PKA-II) isozyme is primarily localized to the cytosol. Because the functions of both PKA-I and PKA-II isozymes in the biochemical events of T lymphocyte activation have not been clearly elucidated, we tested the hypothesis that very early events of normal human T lymphocyte activation are mediated by the PKA-I and/or PKA-II isozyme(s). Fresh normal human T cells and a normal human CD4+ T cell line (GK606) activated with anti-CD3-epsilon and recombinant interleukin 1 alpha (rIL-1 alpha) exhibited a peak six- to sevenfold increase of PKA phosphotransferase activity at 5 min that returned to baseline by 60 min. Similarly, both fresh T cells and the T cell line activated by phorbol myristate acetate and ionomycin demonstrated a peak eightfold increase of PKA activity by 15 min that returned toward baseline by 60 min. Chromatographic separation of the PKA isozymes and quantification of phosphotransferase activities after T cell activation by either agonist pair showed preferential activation of the PKA-I isozyme, resulting in a significant reduction in the ratio of PKA-I to PKA-II isozyme activity from 3.1:1-6.2:1 to 1.1:1-3.2:1. PKA-I isozyme activation resulted in the release of free catalytic (C) subunit, an increase in C subunit phosphotransferase activity, and the phosphorylation of T cell plasma membrane-associated proteins, p14, p17, p20, p21, p38, and p48. However, activation of the PKA-I isozyme did not appear to be required for the transcription of IL-2 mRNA, an event necessary for mitosis. These data indicate that ligand-induced T cell activation is associated with rapid activation of the PKA-I, but not PKA-II, isozyme that results in phosphorylation of plasma membrane-associated proteins. The involvement of the PKA-I isozyme during the very early events of T cell activation suggests that this isozyme may be an antigen- or mitogen-stimulated protein kinase.