Biliary brush cytology is the standard method of sampling a biliary stricture but has a low sensitivity for the detection of malignancy. We have previously shown that minichromosome maintenance (MCM) replication proteins (Mcm2–7) are markers of dysplasia and have utilised these novel biomarkers of growth for the diagnosis of cervical and bladder cancer. We aimed to determine if MCM proteins are dysregulated in malignant pancreaticobiliary disease and if levels in bile are a sensitive marker of malignancy. In 30 tissue specimens from patients with malignant/benign biliary strictures, we studied Mcm2 and -5 expression by immunohistochemistry. Bile samples were also collected prospectively at endoscopic retrograde cholangiopancreatography from 102 consecutive patients with biliary strictures of established (n=42) or indeterminate aetiology (n=60). Patients with indeterminate strictures also underwent brush cytology as part of standard practice. Bile sediment Mcm5 levels were analysed using an automated immunofluorometric assay. In benign biliary strictures, Mcm2 and -5 protein expression was confined to the basal epithelial proliferative compartment – in contrast to malignant strictures where expression was seen in all tissue layers. The percentage of nuclei positive for Mcm2 was higher in malignant tissue (median 76.5%, range 42–92%) than in benign tissue (median 5%, range 0–33%) (P<0.0005), with similar results for Mcm5. Minichromosome maintenance protein 5 levels in bile were significantly more sensitive than brush cytology (66 vs 20%; P=0.004) for the detection of malignancy in patients with an indeterminate stricture, with a comparable positive predictive value (97 vs 100%; P=ns). In this study, we demonstrate that Mcm5 in bile detected by a simple automated test is a more sensitive indicator of pancreaticobiliary malignancy than routine brush cytology.