Chromosome 9p21 appears to harbor a tumor suppressor gene, as evidenced by deletions in this region in a variety of human primary tumors and cell lines. To map the deletion at 9p21 in bladder tumors, we analyzed DNA from 28 tumor and normal pairs at five microsatellite markers that flank the region occupied by the putative tumor suppressor genes p16 and p15. Loss of heterozygosity (LOH) at the markers human interferon (HIFN) alpha and D9S171, which are adjacent to the p15 and p16 loci, was detected in 41% and 33%, respectively, of informative cases of bladder tumors. No sequence mutations were detected in exons 1 or 2 of either p15 or p16 in any of the bladder tumors. Three sequence-tagged site markers in the region bordered by HIFN alpha and D9S171 were used to further map the deleted region by multiplex polymerase chain reaction with the HIFN gamma maker (on chromosome 12) as a control for amplification. Six of 11 tumors with LOH at surrounding markers had homozygous deletions of the marker c5.1, which is located within the p16 gene; and two tumors appeared to have homozygous deletions within p15 (RN1.1) but not p16 (c5.1). A recently identified microsatellite marker, p16-CA-1, located 16 kb distal to p16, proved valuable in defining the minimal deletion involved in these bladder tumors. Five tumors exhibited homozygous deletions of this marker but not HIFN alpha and two tumors showed LOH at this marker and homozygous deletion of p16. Although these data could not be used to identify p16 or p15 as the definitive tumor suppressor gene in this region that is involved in bladder carcinogenesis, they suggest that homozygous deletion is a common mechanism of loss of tumor suppressor gene function in this region.