In the urinary bladder, nitric oxide (NO) influences efferent and afferent neurotransmission (Alfieri et al., 2001; Giglio et al., 2005). In patients suffering from interstitial cystitis, afferent pathways are sensitized and the release of NO is associated with the condition. In cyclophosphamide (CYP)-induced cystitis in the rat, an upregulation of muscarinic M5 receptors occurs, particularly in the urothelium, together with an increase in endothelial NOS expression in the submucosa/mucosa (Giglio et al., 2005). The current study aimed to explore the tissue origin of NO in the normal and inflamed urinary bladder and to compare in vitro findings with effects in in vivo conditions. With local ethics committee approval, male rats were either pre-treated with saline or CYP (100 mg/kg) intraperitoneally and 48-60 h later, the bladder function was examined in in vitro and in in vivo (anaesthetized (Ketalar 50 mg/kg combined with Domitor 0.3 mg/kg i.p.) and conscious rats) experiments. In the CYP-pretreated rats, the contractile in vitro response to methacholine was substantially reduced in muscle strip preparations from the bladder body but not in strips from the trigonum. In normal detrusor preparations, the nitric oxide synthase inhibitor N-ω-nitro-L-arginine (L-NNA; 10-4 M) increased the cholinergic contractile effect, while it had no effect on trigonal muscle strips. In inflamed body preparations, the L-NNA effect was substantially larger than in the normal preparations. The administration of 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; 10-8 M) enhanced the carbachol-induced contractile responses of inflamed strips but not in normal strips. In anaesthetized rats, methacholine induced smaller contractions (i.e. bladder pressure increases) in inflamed bladders than in controls. The removal of the urothelium did not affect the cholinergic contractions in normal bladders, while it enhanced them in inflamed bladders. The NO synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME; 30mg/kg i.v.) caused the methacholine-evoked contractions to be of similar magnitude in urothelium-denuded inflamed bladders as in intact inflamed bladders. In conscious rats, assessing the urodynamics in a metabolic cage, voiding volumes were significantly lower in CYP pre-treated than in saline pre-treated rats. Neither 4-DAMP nor L-NAME had any effect in the normal rats. In CYP pre-treated rats, both 4-DAMP and L-NAME significantly increased voiding volumes. Conclusions: In CYP-induced cystitis, the cholinergic function of the bladder is altered in the bladder body but not in the trigonum. The changes of the cholinergic effects, involving the production of NO presumably occur in the urothelium. The in vitro findings are supported by in vivo findings in anaesthetized as well as in conscious rats.