To identify the location of one or more of the putative tumor suppressor genes (TSG) on chromosome arm 4q that may be involved in hepatocellular carcinoma (HCC), we examined 96 primary HCCs for their patterns of allelic loss at 39 microsatellite marker loci distributed along this chromosome arm. Allelic loss at one or more loci was observed in 71 (74%) HCCs. Detailed deletion mapping identified two distinct commonly deleted regions; one was located within a 1-cM interval flanked by D4S1534 and D4S2929 at 4q21-22, the other in the 1-cM interval flanked by D4S2921 and D4S2930 at 4q35. Of the tumors for which clinical data were available, allelic loss at 4q35 was more frequent in poorly or moderately differentiated tumors than in well-differentiated tumors (3/15, 20%, vs. 14/21, 67%, P = 0.008); in tumors larger than 2 cm in size (2/11, 18%, vs. 34/62, 55%, P = 0.046); and in tumors that arose from liver cirrhosis as opposed to HCCs arising from chronic hepatitis (25/42, 60%, vs. 9/27, 33%, P = 0.048). The association of allelic losses on 4q35 with larger tumor size and aggressive histological type implies that loss or inactivation of TSG located within the 1-cM interval of 4q35 identified here contribute to progression of HCCs.