Cutaneous pigmentation is determined by the amounts of eumelanin and pheomelanin synthesized by epidermal melanocytes and is known to protect against sun-induced DNA damage. The synthesis of eumelanin is stimulated by the binding of alpha-melanotropin (alpha-melanocyte-stimulating hormone) to the functional melanocortin 1 receptor (MC1R) expressed on melanocytes. The human MC1R gene is highly polymorphic and certain allelic variants of the gene are associated with red hair phenotype, melanoma and non-melanoma skin cancer. The importance of the MC1R gene in determining skin cancer risk led us to examine the impact of specific polymorphisms in this gene on the responses of human melanocytes to alpha-melanotropin and UV radiation. We compared the ability of human melanocyte cultures, each derived from a single donor, to respond to alpha-melanotropin with dose-dependent stimulation of cAMP formation, tyrosinase activity and proliferation. In each of those cultures the MC1R gene was sequenced, and the eumelanin and pheomelanin contents were determined. Human melanocytes homozygous for Arg160Trp, heterozygous for Arg160Trp and Asp294His, or for Arg151Cys and Asp294His substitutions, but not melanocytes homozygous for Val92Met substitution, in the MC1R demonstrated a significantly reduced response to alpha-melanotropin. Additionally, melanocytes with a non-functional MC1R demonstrated a pronounced increase in their sensitivity to the cytotoxic effect of UV radiation compared with melanocytes expressing functional MC1R. We conclude that loss-of-function mutations in the MC1R gene sensitize human melanocytes to the DNA damaging effects of UV radiation, which may increase skin cancer risk.