A body of indirect evidence has suggested the involvement of reactive oxygen species (ROS) in tumor promotion. However, direct evidence for the involvement of in situ generated ROS in tumor promotion is lacking at present. This study provides the first in situ evidence for the involvement of ROS in stage I tumor promotion. Earlier we have shown that parenteral administration of Photofrin - II (a mixture of porphyrins) to mice followed by their exposure to visible light generates ROS. In this study we further provide E.S.R spectral evidence that both O2.- and (.OH) radicals are generated during tissue photosensitization. The free radicals/ROS generation is followed by the development of cutaneous inflammation which is maximum at six hours after photosensitization and develops in a dose dependent manner. The epidermal myeloperoxidase activity which represents neutrophil infiltration is also increased more than 160% of the control value. The histopathology of skin tissues of 7,12 dimethyl benz(a)anthracene initiated mice receiving multiple treatments of Pf - II and light for a period of four weeks indicates pronounced epidermal hyperplasia, glandular hyperplasia, dark basal keratinocytes induction characterized by the high uptake of the dye and frequent neutrophil infiltrations. Our data indicate that ROS generated in situ as a result of porphyrin - mediated cutaneous photosensitization results in the development of changes characteristic of stage I tumor promotion in murine skin. © 1995 by Academic Press, Inc.