We have reported that both dehydroepiandrosterone (DHEA) and dexamethasone (Dexa) directly activate PKC. In this study, we investigated the effects of these hormones on conventional PKC (cPKC) and atypical PKC (aPKC). DHEA and Dexa directly activated PKC beta and PKC zeta to the same degree. In rat adipocytes, DHEA and Dexa activated endogenous immunoprecitable PKC zeta to 246 and 164%, respectively, from basal level (100%). In adipocytes, 5 min treatment with DHEA increased phosphatidylinositol 3-kinase (PI 3-kinase) activity in immunoprecipitate with antiphosphotyrotyrosine antibody to 235%. Preincubation with wortmannin, myristoylated PKC zeta pseudosubstrate, but not with Go6976, abolished DHEA-induced 2-deoxyglucose (DOG) uptake. cPKC inhibitors prevented Dexa-induced insulin resistance. Moreover, DHEA and Dexa increased DOG uptake to 330 and 220%, respectively, in adipocytes overexpressed with wild-type PKC zeta, but not in those overexpressed with dominant negative. These results indicate that DHEA and Dexa activate both cPKC and aPKC, and Dexa-induced cPKC activation may lead to insulin resistance. In contrast, DHEA may mimic or enhance insulin action via PI 3-kinase and aPKC.