DNA ploidy analysis of 22 lesions arising from the intrahepatic and extrahepatic biliary systems and gallbladder was performed on Feulgen- stained 5-μm sections from archival paraffin-embedded tissue on an image analyzer (CAS-200). The cases included intrahepatic cholangiocarcinoma (n = 6), extrahepatic bile duct carcinoma (n = 5), carcinoma of the gallbladder (n = 11), and appropriate controls. All malignancies were stage III and IV adenocarcinomas with the exception of 1 stage II moderately differentiated gallbladder adenocarcinoma. No correlation with ploidy and stage could be made, most likely because of the advanced stage of the tumors at the time of presentation. When DNA ploidy was compared with the grade of tumor, the 3 well-differentiated adenocarcinomas (2 cholangiocarcinomas, 1 bile duct carcinoma) were predominantly diploid; however, diploid peaks were also found in moderately differentiated adenocarcinomas of all 3 sites and in a poorly differentiated adenocarcinoma of the extrahepatic bile ducts. Finally, 15 of 22 (68%) of cases showed only aneuploid populations. Multiple populations were observed in 19 of the 22 cases; this finding may reflect intratumoral heterogeneity and correlate with the advanced stage and aggressive nature of malignancies of the gallbladder and biliary system.