Loss of heterozygosity of chromosome region 9p21 occurs commonly and early in sporadic melanoma, suggesting the involvement of a tumor suppressor gene at this locus in the pathogenesis of this neoplasm. Although germline mutations and deletions of the p16INK4 gene located at 9p21 have been reported in familial melanoma, the relative contributions of mutation and deletion in sporadic melanoma are at present unclear. In this study, we investigated 26 cases of sporadic cutaneous melanoma (14 of which demonstrated loss of heterozygosity at 9p21) for mutations of p16INK4. One tumor with allelic loss of 9p contained a CC-->TT mutation at codons 57/58, altering an arginine to a stop codon, consistent with bi-allelic inactivation of p16INK4 in this case. No mutations were identified in any of the other melanomas, or in one benign intradermal nevus with atypical features and two Spitz nevi that also showed loss of heterozygosity of 9p. The inactivation of both copies of p16INK4 in the one case of melanoma adds support to the theory that p16INK4 is important in the development of sporadic cutaneous melanoma, although allelic loss or other methods of inactivation of p16INK4 rather than point mutation appears to be numerically more important. The low frequency of mutation of p16INK4 in cases of sporadic melanoma with loss of heterozygosity of 9p is, however, also consistent with there being another tumor suppressor gene near this locus that is involved in some cases of sporadic melanoma.