Critical roles played by some protein kinases in neoplastic transformation and progression provide a rationale for developing selective, small-molecule kinase inhibitors as antineoplastic drugs. Protein kinase Cepsilon (PKCepsilon) is a rational target for cancer therapy, because it is oncogenic and prometastatic in transgenic mouse models. PKCepsilon is activated by sn-1,2-diacylglycerol (DAG). Attempts to develop selective PKCepsilon inhibitors that block activation by DAG or compete with ATP have not yet met with success, suggesting a need for new strategies. We previously reported that cystamine and a metabolic cystine precursor inactivate PKCepsilon in cells in a thiol-reversible manner. In this report, we first determined that PKCepsilon became resistant to inactivation by disulfides when Cys452 was replaced with alanine by site-specific mutagenesis of human PKCepsilon or a constitutively active PKCepsilon mutant. These results showed that the disulfides inactivated PKCepsilon by thiol-disulfide exchange, either upon Cys452 S-thiolation or by rearrangement to an intra-protein disulfide. Mass spectrometric analysis of peptide digests of cystamine-inactivated, carbamidomethylated PKCepsilon detected a peptide S-cysteaminylated at Cys452, indicating that Cys452 S-cysteaminylation is a stable modification. Furthermore, PKCepsilon inactivation by N-ethylmaleimide was Cys452 dependent, providing corroborative evidence that PKCepsilon inhibitors can be designed by targeting Cys452 with small molecules that stably modify the residue. Cys452 is an active site residue that is conserved in only 11 human protein kinase genes. Therefore, the PKCepsilon-inactivating Cys452 switch is a rational target for the design of antineoplastic drugs that selectively inhibit PKCepsilon.