Organic peroxides, widely used in the chemical and pharmaceutical industry are considered to be one of the key factors contributing to skin tumor promotion via free radical production. In vitro experiments have demonstrated metal-catalyzed formation of alkoxyl, alkyl, and aryl radicals in keratinocytes incubated with cumene hydroperoxide. The present study investigated in vivo free radical generation in lipid extracts of mouse skin exposed to cumene hydroperoxide. The ESR spin-trapping was used to detect the formation of alpha-phenyl-N-tert-butylnitrone (PBN) radical adducts, following intradermal Injection of 180 mg/kg PBN. After topical exposure (30 min), cumene hydroperoxide (12 mmole/kg) induced free radical generation in the skin of female Balb/c mice kept for 10 weeks on vitamin E-deficient diets. In contrast, hardly discernible radical adducts were detected when cumene hydroperoxide was applied to the skin of mice fed a vitamin E-sufficient diet. Total antioxidant reserve, levels of GSH, ascorbate and vitamin E decreased 34%, 46.5% 27% and 98%, respectively, after mice were kept for 10 weeks on vitamin E-deficient diet. PBN adducts detected by ESR in vitamin E- deficient mice provide direct evidence for in vivo free radical generation in the skin after exposure to cumene hydroperoxide.