The present study examines the effect of tobacco smoking on the expression of cyclooxygenase (COX)-2 gene, COX enzymatic activity and prostaglandin (PG) synthesis in urothelial mucosal tissues from patients with bladder cancer and from normal individuals. The detection frequency of COX-2 mRNA was 2-fold higher in bladder cancer patients compared to controls and it was accompanied by a significantly increased COX enzymatic activity and PGE2 synthesis (p < 0.05). Smokers, in both control and patients groups, had higher COX-2 expression, COX activity, and PGE2 synthesis compared to the nonsmokers (p < 0.05). The number of cigarettes smoked in the cases, but not controls, correlated well with COX enzymatic activity (r = 0.42, p = 0.016). The observed over-expression of COX-2 gene in human urinary bladder and the concomitant increases in PG synthesis may explain, at least in part, the mechanism by which cigarette smoking influences the development of urothelial neoplasia.