While melanomas are resistant to the cytotoxic effects of radiotherapy, little is known about the molecular mechanisms underlying this intrinsic resistance. Here, we describe the utilization of retroviral insertional mutagenesis to facilitate the analysis of genetic changes that are associated with radioresistance in human melanoma. A radial growth phase human melanoma cell line, WM35, was infected with a replication-defective amphotropic murine retrovirus and subsequently selected for X-ray radiation-resistant variants. Several radiation-resistant clones were independently isolated and characterized. Interestingly, these clones also displayed resistance to ultraviolet radiation and to the chemotherapeutic drug cis-diamminedichloroplatinum(II) (CDDP). By Northern and Western analyses, we showed that the expression of DOPAchrome tautomerase (DCT), also known as tyrosinase-related protein 2 (TYRP2), an enzyme that functions in eumelanin synthesis, was significantly elevated in the radiation-resistant clones relative to the parental WM35 cells. Moreover, the levels of DCT in a variety of human melanoma cell lines correlated with their relative levels of radioresistance and the enforced expression of DCT conferred increased resistance to UV(B) treatment. An analysis of stress signaling induced by radiation as well other cytotoxic stressors showed that resistance associated with DCT overexpression applied specifically to treatments that activate the ERK/MAPK pathway. Indeed, DCT overexpression in a melanoma cell line resulted in increased ERK activity. Moreover, ectopic expression of dominant-active MEK in this melanoma cell line conferred UV(B) resistance suggesting that the ERK/MAPK pathway downstream of DCT may play a critical role in radiation and drug resistance. Overall, given that each gamma- and UV(B)-resistant cell line also exhibited resistance to CDDP and that CDDP-resistant clones showed increased resistance to UV(B) irradiation, these results suggest a common mechanism underlying radio- and chemoresistance, which is mediated by DCT expression.