In a preliminary study, we demonstrated a strong association between the concentration of the glycoprotein fibronectin (FN) in human bile fluid and the presence of malignant biliary diseases. We now present the results of measurements of total FN (tFN) and cellular FN (cFN) within a larger group of 71 patients. Bile fluid was collected during routine endoscopic retrograde cholangiography or by transhepatic puncture, respectively, from patients admitted for examination/treatment of biliary obstruction. Determination of tFN in bile was performed using a previously described time-resolved fluorescence immunoassay (TRFIA). For cFN, a newly developed TRFIA, using a specific monoclonal antibody for the EDA epitope of cFN, was applied. Within the noncarcinoma group of patients (n=50), consistently low concentrations of tFN (median = 5 ng/mL) were found. In most of these cases, the corresponding concentrations of cFN were below the detection limit (2.6 ng/mL) of this assay. Highly significantly elevated concentrations were found for both tFN (median = 1,220 ng/mL) and cFN (median = 243 ng/mL) in the carcinoma group (n = 21) in comparison with the noncarcinoma group (P < or = .01). By adopting cutoff values of 60 ng/mL for tFN and >0 ng/mL for cFN, diagnostic sensitivities for carcinoma of the biliary tract of 0.89 and 0.92, and specificities of 0.96 and 0.98, respectively, were computed. FN in bile fluid is suggested as a sensitive, specific, and easily determined marker for differential diagnosis of malignant and benign diseases of the biliary tract.