There is increasing evidence for the generation of reactive oxygen species in skin upon ultraviolet exposure, but little is known about their pathophysiologic relevance in human skin in vivo. We hypothesized that chronic and acute photodamage is mediated by depleted antioxidant enzyme expression and increased oxidative protein modifications. Biopsies from patients with histologically confirmed solar elastosis, from non-ultraviolet-exposed sites of age-matched controls, and from young subjects were analyzed. To evaluate the influence of acute ultraviolet exposures, buttock skin of 12 healthy subjects was irradiated repetitively on 10 d with a solar simulator and compared intraindividually to non-ultraviolet-treated contralateral sites. The antioxidant enzymes catalase, copper-zinc superoxide dismutase, and manganese superoxide dismutase were investigated by immunohistochemistry. Protein carbonyls were analyzed by immunohistochemical and immunoblotting techniques in human skin and in cell models. Whereas overall expression of antioxidant enzymes was very high in the epidermis, low baseline levels were found in the dermis. In photoaged skin, a significant depletion of antioxidant enzyme expression was observed within the stratum corneum and in the epidermis. Importantly, an accumulation of oxidatively modified proteins was found specifically within the upper dermis of photoaged skin. Upon acute ultraviolet exposure of healthy subjects, depleted catalase expression and increased protein oxidation were detected. Exposures of keratinocytes and fibroblasts to ultraviolet B, ultraviolet A, and H2O2 led to dose-dependent protein oxidation and thus confirmed in vivo results. In conclusion, the correlation between photodamage and protein oxidation was demonstrated for the first time, which hence may be a relevant pathophysiologic factor in photoaging.