Alterations in the repeat length of microsatellites have been identified recently in tumors arising in patients with hereditary nonpolyposis colon cancer and in several human sporadic tumors. We examined 40 sporadic melanomas and their corresponding nontumorous skin for microsatellite instability (MSI) and loss of heterozygosity (LOH) at chromosomes 2q, 3p25-26, 5q11.2-13.3, 5q21, 6q27, 9p21, 9p22-pter, 17p12, 17p12-p11.1, and 18q23. Specific loci were amplified by polymerase chain reaction, electrophoresed on polyacrylamide gels, transferred onto nylon membranes, and hybridized with 33P-end-labeled oligonucleotides. MSI was observed in eight of 40 (20%) melanomas at one of 10 loci examined. LOH was found at chromosome region 9p21 in 40%, at 9p22 in 22%, and at 17p in 13% of the informative cases. Comparison between clinicopathologic features of patients with and without MSI revealed no obvious differences. LOH at 9p21 was observed only in lesions greater than 1.5 mm in depth, suggesting that it does not represent an early event in sporadic melanoma. Our results indicate that 1) MSI is a genetic alteration in a proportion of sporadic melanoma, which may reflect a defect in genes involved in DNA replication fidelity; and 2) LOH at chromosome region 9p21 is a significant event in sporadic melanoma. The latter finding further supports the hypothesis that the 9p21 region may contain one or more tumor suppressor genes (e.g., MTS1/CDNK2) involved in the pathogenesis of melanoma.