The presence of ozone (O 3 ) in photochemical smog is an important health concern. We hypothesized that the stratum corneum (SC), as the outermost skin layer and the permeability barrier of the skin, represents a sensitive target for O 3 -induced oxidative stress. To test this hypothesis, SKH-1 hairless mice were anesthetized and exposed for 2 h to O 3 by using two strategies: (i) single exposures to 0 (n = 12), 1 (n = 4), 5 (n = 4), and 10 (n = 4) ppm; and (ii) repeated daily exposures to 0 ppm (controls; n = 4) and 1 ppm (n = 4) for six consecutive days. New techniques based on the removal of SC by tape stripping were used to analyze the biologic effects of O 3 with respect to vitamin E depletion and lipid peroxidation. SC tissue was extracted from the tape and immediately analyzed by HPLC for vitamin E and malondialdehyde (MDA) concentrations. After in vivo exposure to increasing O 3 doses, vitamin E was depleted and MDA formation was increased, both in a dose-dependent manner. Remarkably, repeated low-level O 3 exposures resulted in cumulative oxidative effects in the SC: As compared with O 3 exposures of 0 ppm (α-tocopherol, 8.95 ± 1.3 pmol per mg; γ-tocopherol, 3.00 ± 0.3 pmol per mg; MDA, 3.69 ± 0.3 pmol per mg), vitamin E was depleted (α-tocopherol, 2.90 ± 0.6 pmol per mg, p < 0.001; γ-tocopherol, 0.5 ± 0.1 pmol per mg, p < 0.001) and MDA levels were increased (4.5 ± 0.2; p < 0.01). This report demonstrates the unique susceptibility of the SC to oxidative damage upon exposure to O 3 .