We have previously demonstrated the existence of a melanoma tumor suppressor gene(s) on the long arm of chromosome 11 through suppression of tumorigenicity assays. Although loss of heterozygosity studies also support this finding, only a large critical region (44 cM) has been identified to date on 11q22-25. To further localize a tumor suppressor gene(s) within this region, we have now generated and characterized nine melanoma microcell hybrids, each retaining an introduced fragment of 11q. Of the nine hybrids, four were suppressed for tumor formation in nude mice, while five formed tumors at the same rate as the parental melanoma cell line (UACC 903). Molecular analysis of the hybrids with 118 microsatellite markers narrowed the location of a putative suppressor gene to a small (< or =2 Mb) candidate region on 11q23 between the markers D11S1786 and D11S2077 and within the larger region frequently deleted in melanoma tumors and cell lines. While multiple tumor suppressor genes are likely to reside on 11q22-25, the presence of this region in all four suppressed hybrids supports the simplest model that a single locus is responsible for the suppressed phenotype observed in UACC 903.