The INK4a/ARF locus encodes 2 cell cycle regulatory proteins: p16 and p14(ARF). P16 inhibits the activities of cdks, which maintain the retinoblastoma protein (pRb) in its active hypophosphorylated state. P14(ARF) blocks MDM2-induced p53 degradation and transactivational silencing. In this study, we investigated the expression of p16 and p14(ARF) in reference human urothelium and in 51 urothelial carcinomas (UCs) of all stages and grades, by reverse transcription-polymerase chain reaction (RT-PCR). Patterns of p14(ARF) and p16 expression were compared with each other and then with patterns of p53 and pRb protein expression, respectively, as determined by immunohistochemistry. P14(ARF) and p16 mRNAs were present at low levels or were undetectable in reference urothelia and in most superficial tumors, whereas they were present at high levels in a subset of tumors of advanced stage and high grade. The expression profiles of these 2 mRNAs were correlated in all but 4 cases, indicating that the 2 INK4a products may have nonredundant functions. Forty-six of the 51 tumors (90%) presented changes to or a lack of activation of the p14(ARF)-p53 pathway and were p53 positive (n = 10), p14(ARF) negative (n = 23), or both p53 positive and p14(ARF) negative (n = 13), suggesting that these 2 components of the pathway may be altered or nonactivated. Markedly high levels of p16 mRNA (n = 5) were associated with the absence of pRb expression, with the exception of 1 case in which the p16 gene contained a deletion mutation. A lack of p16 mRNA or low levels of this mRNA were associated with pRb detection in all but 1 case. In invasive UCs, the p16-pRb pathway, the p14(ARF)-p53 pathway, or in many cases both pathways were altered or not activated, demonstrating the involvement of these pathways in invasive bladder tumorigenesis.