Abstract Background & Aims : Digital image analysis (DIA) allows quantification of nuclear DNA content and may help distinguish benign and malignant strictures of the biliary tract. Methods : One hundred ten consecutive patients undergoing endoscopic retrograde cholangiography for suspicious biliary tract strictures were enrolled in a prospective study comparing the accuracy of DIA and routine cytology (RC). Standard brush cytology sampling was performed twice by using 2 cytology brushes per patient. Both brushes were fixed in a single-specimen vial. Each specimen was formed into 1 pellet, and the sample was equally divided for evaluation by DIA and RC. DNA histograms were generated for ploidy analysis. The DIA criterion for malignancy was demonstration of aneuploidy. Results : Two patients had inadequate samples obtained for DIA analysis, 7 benign patients were excluded because of inadequate follow-up of less than 75 days, and 1 patient was lost to follow-up to clarify malignant versus benign disease. Of the remaining 100 patients, 56 strictures were malignant and 44 were benign. The sensitivities of DIA and RC were 39.3% and 17.9%, respectively ( P = 0.014). The specificities of DIA and RC were 77.3% and 97.7%, respectively ( P = 0.003). The accuracy of DIA (56.0%) was equivalent to RC (53.0%). Conclusions : DIA is a valuable adjunct to RC for detecting malignant strictures of the biliary tract.