Cigarette smoking is an independent risk factor for inducing vasculogenic erectile dysfunction (ED). NO has been demonstrated to be the principal mediator of cavernous smooth muscle relaxation and penile erection. Therefore, we examined whether or not enzyme activities and factors involved in NO generation pathway are affected in rabbit corpus cavernosum following administration of nicotine-and tar-free cigarette smoke extract (CSE). CSE was prepared by bubbling a stream of cigarette smoke into phosphate buffered saline. CSE was injected subcutaneously to adult male rabbits once a day for 5 weeks. In CSE group, significantly decreased cyclic GMP production as a marker of NO generation was associated with the attenuated overall NOS activity, enhanced arginase activity, accumulation of endogenous NOS inhibitors such as monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), and decreased dimethylarginine dimethylaminohydrolase (DDAH) activity as an metabolizing enzyme of endogenous NOS inhibitors. Neuronal NOS (nNOS) and DDAH I proteins expression were decreased without altering endothelial NOS (eNOS) expression, while arginase I expression was up-regulated. These results suggest that the impaired NO production would result from blunted NOS activity, which is possibly brought about by the down regulation of nNOS protein, accumulation of endogenous NOS inhibitors and enhanced arginase activity together with up-regulation of arginase I protein in cavernous tissue. The impaired DDAH activity due to decreased expression of DDAH I protein would result in an accumulation of endogenous NOS inhibitors with CSE. These alterations may be relevant to inducing the erectile dysfunction following CSE.