The specific roles of nitric oxide (NO) synthases (NOSs) in bladder smooth muscle remain to be elucidated. We examined the roles of NOSs in beta- adrenoceptor (AR)mediated bladder relaxation. Male mice (C57BL6) deficient of neuronal NOS [nNOS-knockout (KO)], endothelial NOS (eNOS-KO), neuronal/endothelial NOS (n/eNOS-KO), neuronal/endothelial/ inducible NOS (n/e/iNOS-KO), and their controls [wild-type (WT)] were used. Immunohistochemical analysis was performed in the bladder. Then the responses to relaxing agents and the effects of several inhibitors on the relaxing responses were examined in bladder strips precontracted with carbachol. Immunofluorescence staining showed expressions of nNOS and eNOS in the urothelium and smooth muscle of the bladder. Isoproterenol-induced relaxations were significantly reduced in nNOS-KO mice and were further reduced in n/eNOS-KO and n/e/iNOS-KO mice compared with WT mice. The relaxation in n/e/iNOS-KO mice was almost the same as in n/eNOS-KO mice. Inhibition of Ca-21 - activated K-1 (K-Ca) channel with charybdotoxin and apamin abolished isoproterenol-induced bladder relaxation in WT mice. Moreover, direct activation of K-Ca channel with NS1619 caused comparable extent of relaxations among WT, nNOS-KO, and n/ eNOS- KO mice. In contrast, NONOate (a NO donor) or hydrogen peroxide (H2O2) (another possible relaxing factor from eNOS) caused minimal relaxations, and catalase (H2O2 scavenger) had no inhibitory effects on isoproterenol-induced relaxations. These results indicate that both nNOS and eNOS are substantially involved in beta-AR-mediated bladder relaxations in a NO-or H2O2-independent manner through activation of K-Ca channels.