Prolonged cell proliferation in response to irritation by calculi may itself evoke malignant transformation of the urothelium. However, the molecular mechanisms underlying this process are still unknown. The aim of the present study was to investigate cell cycle regulatory mechanisms in bladder carcinogenesis induced by bladder calculi. Six-week-old Wistar rats were consecutively fed a diet containing 5% terephthalic acid (TPA), 5% TPA plus 4% sodium bicarbonate (NaHCO(3)), 4% NaHCO(3), or basal diet for 48 weeks. Animals were killed at weeks 12, 24, and 48. Treatment with 5% TPA caused high incidences of bladder calculi, preneoplastic lesions, and neoplastic lesions. Immunohistochemical examination revealed overexpression of cyclin D1, cyclin-dependent kinase 4 (Cdk4), retinoblastoma (Rb), and proliferating cell nuclear antigen (PCNA) in bladder preneoplastic and neoplastic lesions. In contrast, p16 expression was reduced or absent. These results were confirmed by immunoblotting analysis. Quantitation of mRNA by real-time reverse transcription-polymerase chain reaction (RT-PCR) showed a significant increase in cyclin D1 and PCNA mRNA in tumor cells. None of the 16 transitional cell carcinomas (TCCs) had ras mutations as examined by PCR-single strand conformational polymorphism (PCR-SSCP) analysis. These results suggested that deregulation of p16-cyclin D1/Cdk4-Rb pathway, but not oncogenic activation of ras, plays a crucial role in bladder tumorigenesis induced by bladder calculi.