Aims: Photochemotherapy employing psoralens combined with UVA irradiation (PUVA) is a standard therapy for a variety of dermatoses. Psoralens can be administered orally or topically in the form of bath or cream preparations. Recommendations for the time of UVA irradiation are mainly based on the time course of minimal phototoxic doses. However, the time course and depth of skin penetration of psoralens is not well characterized. Methods: We assessed the time course of 8-MOP concentrations in horizontal epidermal and dermal skin sections in 10 patients undergoing oral (n = 3), cream (n = 4) and bath (n = 3) PUVA therapy. Punch biopsies (4 mm) were taken from 'healthy' skin sites. A highly sensitive LC-MS-MS method was employed for 8-MOP analysis. Results: Epidermal concentrations following cream or bath were highest at the end of the application period (time zero) when irradiation is performed. At this time, 8-MOP cream provided significantly higher epidermal concentrations (mean ± s.e. mean 128.0 ± 22.6 pg mm-3; 95% CI: 77.6, 178.4) than oral 8-MOP (27.0 ± 25.3 pg mm-3; 95% CI: 29.3, 83.3 at 1 h; P = 0.025). Conversely, concentrations in the papillary dermis were significantly higher with oral 8-MOP (20.2 ± 3.1 and 16.2 ± 2.2 pg mm-3 at 1 and 2 h, respectively) than with 8-MOP cream (7.1 ± 2.8 and 8.4 ± 2.0 pg mm-3 time zero and 0.5 h, respectively; P = 0.020 and 0.045, respectively) or bath (8.8 ± 3.1 and 7.7 ± 2.2 pg mm-3; P = 0.050 and 0.039, respectively). The observed time courses of 8-MOP concentrations correspond to time courses of photosensitivity found previously with the different treatment modalities. Conclusions: The higher epidermal 8-MOP concentrations found after topical 8-MOP may explain the lower UVA doses needed with the topical route. These results suggest that topical 8-MOP may be superior in patients where the pathology is localized in the epidermis. In sclerosing diseases, which mainly affect the dermis oral PUVA might be advantageous because dermal concentrations are highest with this route of administration.