A method of membrance permeabilization of T lymphocytes with the bacterial cytotoxin streptolysin O has allowed the effect of guanine nucleotide analogues on phosphatidylinositol metabolism and protein kinase C (PKC) activation to be investigated. The data demonstrate that, in permeabilized cells, phosphorylation of the γ subunit of the CD3 antigen can be induced in response to the PKC activator phorbol 12,13-dibutyrate, the polyclonal mitogen phytohaemagglutinin (PHA) and the stimulatory guanine nucleotide analogue guanosine 5'-[γ-thio]triphosphate (GTP[S]). Application of a pseudo-substrate inhibitor of PKC indicated that CD3γ-chain phosphorylation induced in response to all three agonists was mediated by PKC. PHA and GTP[S] also stimulated inositol phospholipid turnover and inositol phosphate accumulation. The kinetics and concentration-dependence of PHA-induced inositol phospholipid hydrolysis correlated with PHA-induced CD3γ phosphorylation, suggesting that PHA may regulate CD3γ phosphorylation via diacylglycerol produced as a consequence of inositol phospholipid hydrolysis. However, there was an inconsistency in that PHA induced greater (> 200%) levels of inositol phospholipid turnover than did GTP[S], but much weaker (< 50%) levels of CD3-antigen phosphorylation. There was also a discrepancy between GTP[S] effects on phosphatidylinositol turnover and PKC activation, in that the half-maximal GTP[S] concentration for inositol phosphate production and CD3γ-chain phosphorylation was 0.75 μM and 75 μM respectively. Moreover, 10 μM-GTP[S] induced maximal inositol phosphate production, but only 10% of maximal CD3γ-chain phosphorylation. The data are consistent with the idea that other signal-transduction pathways, in addition to those involving inositol phosphate production,exist for the regulation of PKC in T lymphocytes.