We have used polymerase chain reaction (PCR), an amplification procedure, and oligonucleotide hybridization to detect ras gene point mutations in DNA from melanoma tumor samples. Genomic DNA was examined from 40 specimens of melanotic lesions, including benign nevi, primary melanomas, lymph node metastases, and systemic metastases. Adjacent normal skin or peripheral blood was analyzed as control material in 28 cases. ras mutations were detected overall in 25% of malignant tumors. In addition, mutations of all three ras genes were detected. We observed ras mutations in 2 of 4 benign atypical nevi (2 X K12), 4 of 22 primary melanomas (3 X K12, 1 X H12, 1 X N61), and 4 of 14 secondary (5 X K12, 1 X N61) tumors. One with a primary melanoma had concurrent K12 and H12, and two patients with secondary tumors had concurrent K12/N61 and K12 Asp/K12 Val mutations, respectively, making a total of 10 of 40 (25%) patients with ras mutations. This is the first demonstration of K-ras mutations in human melanoma. The presence of K-ras mutations in nevi, putative melanoma precursors, suggests that ras activation may be an early event in melanoma development. No correlation between tumor thickness and the presence of a ras mutation was observed.