Phenotypic modulation of smooth muscle is associated with various pathological conditions including bladder dysfunction. Cytoskeletal dynamics modulate the cell phenotype and were recently shown to be involved in regulation of inducible nitric oxide synthase (iNOS). We tested the hypothesis that the cell differentiation status affects iNOS expression, and that iNOS is preferentially expressed in immature dedifferentiated bladder smooth muscle cells (BSMC). Isolated rat BSMC were put into different stages of differentiation by serum-deprivation on laminin-coated plates in the presence of IGF-1 and by interaction with Rho signalling and actin polymerization. iNOS and smooth muscle-myosin heavy chain (SM-MHC) protein expression were investigated with Western blot analysis. Our results showed iNOS protein in BSMC exposed to interleukin-1 (IL-1; 2 ng/ml) + TNF- (50 ng/ml). Growth of BSMC in serum-free medium on laminin in the presence of IGF-1 increased SM-MHC expression whereas cytokine-induced iNOS was inhibited. Disruption of F-actin with latrunculin B (0.5 µM) potentiated iNOS expression and decreased SM-MHC expression. Rho inhibition with C3 (2.5 µg/ml) increased iNOS expression whereas SM-MHC expression was slightly decreased. Rho-kinase inhibition with Y-27632 (10 µM) mediated a decrease in iNOS and a slight increase in SM-MHC expression. In conclusion, the capacity of BSMC to express iNOS was negatively correlated to differentiation status measured as SM-MHC expression. Actin cytoskeletal dynamics and Rho signalling are involved in regulation of cytokine-induced iNOS expression in BSMC. Phenotypic changes and impairment in actin cytoskeleton formation may potentiate cytokine activation and in turn increase nitric oxide production in the bladder during disease.