Distinction of urothelial carcinoma in situ (CIS) from reactive atypia on the basis of morphology alone may be difficult in some cases. Because this distinction is therapeutically and prognostically critical, we attempted to determine if an immunohistochemical panel would help in this differential diagnosis. The immunoprofile of 21 cases of CIS and 25 non-neoplastic urothelia (15 urothelial biopsies with reactive atypia from patients without a history of bladder cancer and 10 normal ureter sections from nephrectomies performed for renal cell carcinoma) was determined using antibodies against cytokeratin 20 (CK20), p53, and CD44 (standard isoform). In the normal urothelium CK20 showed patchy cytoplasmic immunoreactivity in only the superficial umbrella cell layer and CD44 stained only the basal cells. Nuclear immunoreactivity to p53 varied from negative to weak and patchy. Reactive urothelium also showed CK20 immunoreactivity in only the umbrella cell layer in all 15 cases, and p53 nuclear staining was predominantly negative with occasional weak positivity in the basal and parabasal intermediate cells. CD44 was overexpressed in the entire reactive urothelium in 9 cases (60%) or focally positive in intermediate cells in 6 cases (40%). In contrast, CIS showed intense CK20 and p53 positivity (81% and 57%, respectively) in the majority (>50%) of malignant cells. CD44 staining revealed residual basal cells with membranous reactivity in 44% of the cases of CIS; however, the neoplastic cells were immunonegative in all cases. At least one positive immunomarker (CK20 or p53) was abnormally expressed in all cases of CIS. Abnormal expression of CK20 (increased), p53 (increased), and CD44 (decreased) in urothelial CIS, and increased expression of CD44 in reactive atypia allows more confident distinction of urothelial CIS from non-neoplastic urothelial atypias. From a differential diagnosis perspective, use of a panel of all three antibodies with morphologic correlation would be essential.