Interpreting biliary brush cytology (BBC) findings in primary sclerosing cholangitis (PSC) is problematic. In our study, BBC findings and CA19-9 serum levels were evaluated for their effectiveness in diagnosing cholangiocarcinoma in patients with PSC. We reviewed 107 biliary brushings from 51 patients with PSC and concurrent CA19-9 levels between January 1995 and March 2004 at the University of Washington Medical Center, Seattle. A portion of the brushings were evaluated and scored according to specific cytologic criteria; statistical analysis showed which criteria were most predictive in diagnosing malignancy: nuclear/cytoplasmic ratio, prominent nucleoli, nuclear membrane irregularities, and discohesion were significant predictive features. Sensitivity and specificity of BBC were 62.5% and 100%, respectively. Sensitivity and specificity of CA19-9 at a cutoff of 186 IU/mL were 100% and 94%, respectively. BBC is a specific and relatively sensitive method of detecting cholangiocarcinoma, even in patients with PSC, especially when certain cytomorphologic features are identified. Combining biliary cytology and CA19-9 levels might have an important diagnostic role in PSC.