The short arm of chromosome 3 is considered to harbor one or more of the tumor suppressor genes taking part in the genesis of renal cell carcinoma (RCC). To define the localization of such putative tumor suppressor gene(s), we studied specific allelic loss on chromosome 3p by using 84 samples of RCC with nine microsatellite markers. We defined two commonly deleted regions in 3p14.3-p21.2: (1) region A, a 2-cM region between D3S1313 and D3S1592, and (2) region B, a 2-cM region between D3S1581 and D3S1289. The most frequent loss of heterozygosity was observed at D3S1067 (33 of 59, 55.9%), which is within region A. We further focused on region A and constructed a yeast artificial chromosome (YAC) contig and found that one YAC clone, which was 700-kb in size, harbored the entire region A. Using cosmid clones isolated from this contig, we also performed fluorescence in situ hybridization analysis and found that two of the tumors were homozygously deleted in this region. Our results strongly suggest the existence of a tumor suppressor gene in this region.