Previous studies have shown that expression of cytokeratin 20 (CK20), a constituent of intermediate filaments, is increased in malignant versus benign urine samples. To evaluate whether immunocytochemical staining of CK20 on archived urine slides could be used as a potential adjunct marker for triage of atypical urine cytology, we analyzed a total of 77 archived urine slides obtained from a spectrum of patients with various risks of developing urothelial carcinoma. These patients were divided into four groups on the basis of initial urine cytologic results and subsequent follow-up biopsy findings; group 1 had negative results in both evaluations, whereas the results in group 4 were positive for both cytology and biopsy. Groups 2 and 3 had a diagnosis of atypical urine cytology; however, patients in group 3 had a positive follow-up biopsy, and patients in group 2 did not. The Papanicolaou-stained archived urine slides were destained and then restained immunocytochemically with monoclonal antibody against CK20. With 5% positively stained nonumbrella cells as a threshold, CK20 was positive in 94.4% of group 3 or 4 patients. In contrast, CK20 was positive in 27.3% of group 2 patients and in 10.5% of group 1 patients. The overall sensitivity and specificity for CK20 for the detection of urothelial carcinoma in this population of patients were 94.4% and 80.5%, respectively. This study demonstrated that immunocytochemical analysis of CK20 on archived urine slides could be used to triage atypical urine cytology into low- and high-risk categories and that CK20 might be a simple and useful early detection marker for urothelial carcinoma.