To evaluate skin susceptibility to ozone (O3) and to localize possible oxidative damage within the skin layers, hairless mice were exposed to 10 ppm O3 or air (0 ppm O3) for 2 h. The mice were euthanized, the skin removed and frozen. Three skin layers (upper epidermis, lower epidermis/papillary dermis, and dermis) were separated, antioxidant concentrations (alpha-tocopherol and ascorbic acid) and the lipid peroxidation product malondialdehyde (MDA) measured. In the upper epidermis, O3 significantly depleted alpha-tocopherol (22%; p < .05) and ascorbic acid (55%; p < .01). These antioxidants were unchanged by O3 in the lower skin layers. More remarkably, MDA increased ten-fold in the upper epidermis (p < .001) and two-fold in the lower epidermis/papillary epidermis (p < .05); it was unchanged in the dermis. Thus, exposure to O3 in vivo depletes ascorbic acid and alpha-tocopherol and strongly induces lipid peroxidation in skin. High MDA concentrations measured in the upper epidermis suggest that O3 reacts directly with fatty acids on the skin surface layers. These results further suggest that chronic exposure to lower O3 concentrations found in urban smog could potentially have implications for skin health.