Melanocytic transformation is thought to occur by the sequential accumulation of genetic alterations. Evidence implicating human chromosomes as a site for a gene(s) involved in melanoma suppression comes from studies of LOH [loss of heterozygosity], cytogenetics and biologic reversion of tumorigenicity following the introduction of a normal chromosome 6 by microcell-mediated chromosome transfer (Trent et al., 1990). Using a tumorigenic melanoma cell line (UACC 903) and a chromosome-6 suppressed melanoma subline [UACC 903 (+6)], we have isolated a series of genes uniquely expressed in the suppressed subline. A modified PCR-based cDNA subtraction technique was used to generate subtracted cDNA sublibraries for both the parental and (+6) suppressed cells. A total of 32 randomly selected clones from the suppressed sublibrary were isolated and examined, with 24 detecting a transcript by Northern analysis. Of these 24 clones, 21 (88%) demonstrated elevated expressed by Northern analysis in the suppressed subline relative to the tumorigenic parental cell line. In 6/21 differentially expressed clones (29%), expression was exclusive to the suppressed subline. Partial sequence analysis and database searching of these clones indicated that 5/6 were novel with one representing a previously characterized gene. Chromosomal localization of the five novel clones was performed following PCR amplification of a human/rodent somatic cell hybrid mapping panel or fluorescent in situ hybridization. One cDNA (termed AIM1) was localized to a band-region of chromosome 6 frequently deleted in melanomas (6q21). This novel approach should facilitate the identification of genes whose expression is causally related to the suppressed phenotype.