We immunochemically studied p16(INK4a) expression in 116 urine cytologic samples and compared results with 190 histologic samples. The cytologic samples were classified into 4 groups: 1, mild cellular atypia; 2, moderate cellular atypia; 3, severe cellular atypia; and 4, malignancy. Overexpression of p16(INK4a) was detected in none of 32 cases in group 1, 8 (16%) of 50 cases in group 2, 5 (42%) of 12 cases in group 3, and 11 (50%) of 22 cases in group 4. In addition, by histologic analysis, p16(INK4a) overexpression was not detected in nonneoplastic urothelium, except for a few cases of reactive atypia, but it was detected in about 50% of urothelial carcinomas. In particular, a high incidence (16/20 [80%]) of p16(INK4a) overexpression in high-grade carcinomas was noted in cytologic samples. Immunocytologic analysis of p16(INK4a) expression in cytologic samples is a useful ancillary tool for detection of urothelial carcinoma with infiltrating potential.