The purpose of the study was to analyze endogenous nitric oxide (NO) formation and NO-synthase (NOS) gene expression in the urinary bladder from patients with urinary bladder cancer and to investigate the relationship between local NO formation, treatment with Bacillus Calmette Guerin (BCG) and clinical stage in bladder cancer patients. One hundred and three patients with bladder cancer were studied. Endogenous formation of NO was measured in 72 patients, including 6 patients with BCG treated bladder cancer and 6 tumor free control subjects. iNOS expression was analyzed at transcriptional and protein level in biopsies from 31 patients with bladder cancer by real time polymerase chain reaction (PCR) and Western blot (WB), respectively. Three patients in this group had received BCG treatment. Eight biopsies from normal bladder served as control for PCR and WB analysis. Patients with carcinoma in situ (CIS) had higher iNOS expression (p<0.01) and NO formation (p<0.01) than control subjects and patients with papillary tumors without concomitant CIS. Markedly increased iNOS expression (p<0.05) and NO formation (p<0.001) were also found in patients treated with BCG as compared to the other groups. In conclusion, the presence of elevated NO concentration and iNOS expression in the urinary bladder from BCG treated patients and patients with CIS further supports the notion that NO may be an important factor in bladder cancer biology and that the BCG effect on superficial bladder cancer may partly be due to stimulation of local NO formation.