Tumorigenesis and tumor progression can be considered an evolutionary process. In order to deduce information on the mutational and selective pressures during melanoma progression we performed microsatellite analysis at 42 autosomal and two X-linked loci in a microdissected primary melanoma and its nine metastases. Loss of heterozygosity at locus D9S259 was the only genetic change observed in all metastases. The pattern of loss of heterozygosity at loci D9S162 and D9S171 within the region of common loss on chromosome 9p21 which encompasses the tumor suppressor gene p16ink4 enabled the distinction of four genetically different tumor cell populations. Three cell lineages showed homozygous loss of the p16ink4 gene, which evolved independently in each tumor cell population within the primary tumor. Additional allele losses could be demonstrated at markers D14S53 and DXS998. The fourth lineage did not demonstrate loss of heterozygosity at loci D9S162 and D9S171 and contained the wild type p16ink4 gene but was characterized by abundant microsatellite instability. The evolutionary approach towards tumorigenesis and tumor progression used in this study thus confirms the role of p16ink4 inactivation for melanoma progression but not for melanoma initiation; it suggests the existence of additional putative tumor suppressor genes located on 9p as well as on the long arm of chromosome 14 and shows that microsatellite instability may represent an alternative pathway of tumor cell evolution in malignant melanoma.