Benzoyl peroxide (BzPO) has been the most widely used topical agent for acne since the 1960s. This is true despite numerous reports that BzPO can enhance the development of carcinomas from murine epidermal papillo-mas. Because activation of protein kinase C (PKC) is considered to mediate cellular responses to other epidermal tumor promotors, we wished to investigate the relationship between BzPO and PKC in cultured human epidermal keratinocytes (NHEK). We assayed (a) direct effects of BzPO on PKC activity in a cell-free system using semipurified human keratinocyte PKC, (b) BzPO effects on the subcellular distribution of PKC, and (c) BzPO modulation of NHEK proliferation and phorbol ester-induced differentiation. NHEK maintained in serum-free media (0.15 mMCa2+) were treated with concentrations of BzPO in acetone from 100 nM to 500 μM, with concentrations of acetone not exceeding 0.1%. No short-term translocation of PKC from cytosol to membrane was observed at any BzPO concentration. BzPO did not downregulate subcellular levels of PKC activity after 24 h of exposure. BzPO did not significantly antagonize phorbol ester-induced inhibition of proliferation or differentiation but did weakly antagonize Ca2+-induced differentiation. Consistent with a PKC-mediated mechanism for Ca2+-induced differentiation, BzPO inhibited both human and murine PKC in a cell-free system. These results suggest that BzPO does not promote malignant conversion through a PKC-dependent mechanism, and in fact, inhibits PKC activity in vitro.