Protein kinase C (PKC) is a family of ten isozymes that play distinct and in some cases opposing roles in cell growth and survival. We recently reported that diamide, a diazene carbonyl derivative which oxidizes thiols to disulfides through addition/displacement reactions at the diazene bond, induces potent GSH-dependent inactivation of several PKC isozymes, including the oncogenic isozyme PKC epsilon, via S-glutathiolation. PKC delta, a pro-apoptotic isozyme, was distinguished by its resistance to inactivation. In this report, we show that PKC-regulatory S-thiolation modifications produced by physiological disulfides elicit opposing effects on PKC delta and PKC epsilon activity. We report that PKC delta is stimulated 2.0-2.5 fold by GSSG, (Cys-Gly)(2) and cystine, under conditions where PKC gamma and PKC epsilon are fully inactivated by cystine, and PKC alpha activity is affected marginally or not at all by the disulfides. Focusing on cystine, we show that DTT quenches cystine-induced PKC delta stimulation and PKC gamma and PKC epsilon inactivation, indicative of oxidative regulation. By analyzing DTT-reversible isozyme radiolabeling by [(35)S]cystine, we demonstrate that PKC gamma, PKC delta and PKC epsilon are each [(35)S] S-cysteinylated in association with the concentration-dependent regulation of isozyme activity by cystine. The restricted reactivity of cystine, together with the effects of DTT and thioredoxin on cystine-induced PKC isozyme regulation reported here, indicate that the cystine-induced PKC-regulatory effects entail isozyme S-cysteinylation. We recently hypothesized that antagonism of tumor promotion/progression by small cellular thiols may involve PKC regulation via oxidant-induced S-thiolation reactions with PKC isozymes. The findings of cystine-induced PKC isozyme regulation by S-cysteinylation reported here offer correlative support to the hypothetical model. Thus, PKC delta, a potent antagonist of DMBA-TPA-induced tumor promotion/progression in mouse skin, is stimulated by S-cysteinylation, PKC epsilon, an important mediator of the tumor promotion/progression response, is inactivated by S-cysteinylation, and PKC alpha, which is not influential in DMBA-TPA-induced tumor promotion/progression, is not regulated by cystine. Furthermore, PKC gamma has oncogenic activity, and S-cysteinylation inactivated PKC gamma and PKC epsilon similarly. These findings provide evidence that S-cysteinyl acceptor-sites in PKC isozymes may offer attractive targets for development of novel cancer preventive agents.