Recent studies in our laboratory indicated that inactivation of a putative tumor suppressor gene on chromosome 9q is likely to be associated with an early step of esophageal carcinogenesis. To further define a region containing the putative tumor suppressor gene, we have examined loss of heterozygosity in 37 esophageal squamous cell carcinomas using 14 microsatellite markers mapped to 9q31-q34.1. Loss of heterozygosity was observed in 30 (81%) of 37 tumors at one or more of the loci examined, and partial or interstitial deletions at 9q31-q34.1 were detected in 13 of these tumors. On the basis of these results, we constructed a detailed deletion map and defined a commonly deleted region between the D9S262 and D9S154 loci at 9q31-q32. The genetic distance between these two loci is estimated to be approximately 4 cM.