BACKGROUND The pre-operative differentiation of tumors of the pancreas, Papilla of Vater and the biliary tract is still unsatisfactory. Tumor marker analysis of the pancreatic juice did not improve the pre-operative diagnosis by a great deal. METHODS Bile from resected gallbladders of patients suffering from carcinomas of the pancreato-biliary system was analysed for CA 19-9, CEA, CA 72-4, CA 125 and AFP concentrations. The results were compared to patients suffering from acute cholecystitis, cholecystolithiasis as well as those suffering from benign tumors of the pancreato-biliary region. RESULTS Extreme high CA 19-9 concentrations were found in bile. Evaluations of the tumor-antigens CA 19-9, CA 72-4 and CEA in gallbladder bile were superior to any serum and pancreatic juice examination with respect to sensitivity and specificity. Observed sensitivities amounted to 100% for patients suffering from bile duct carcinoma (CA 19-9) and papillary carcinoma (CEA) at a specificity of 100%. CA 19-9 showed a sensitivity of 76.5% for pancreatic carcinomas at a specificity of 96.4%. CA 19-9 showed significant differences for the local tumor burden and for the degree of lymph node metastasis. Examination of tumor antigens in the gallbladder results in a high degree of discrimination for malignant and benign lesions of the subhepatic pancreato-biliary system. CONCLUSIONS CA 19-9 must follow a entero-hepatic circulation, since it showed raised bile concentrations (factor: 10(4)) compared to serum analysis. Analysis of CA 19-9, CEA and CA 72-4 gives an opportunity for improvement in the detection of cancers of the pancreato-biliary system. Since the clinical important differentiation of tumors of the head of the pancreas (carcinoma vs. pancreatitis) remains unclear, an improvement by bile analyses must be assumed.