We investigated the potency and the selectivity profile of vardenafil on phosphodiesterase (PDEs) enzymes, its ability to modify cGMP metabolism and cause relaxation of penile smooth muscle and its effect on erections in vivo under conditions of exogenous nitric oxide (NO) stimulation. PDE isozymes were extracted and purified from human platelets (PDE5) or bovine sources (PDEs 1, 2, 3, 4 and 6). The inhibition of these PDEs and of human recombinant PDEs by vardenafil was determined. The ability to potentiate NO-mediated relaxation and influence cGMP levels in human corpus cavernosum strips was measured in vitro, and erection-inducing activity was demonstrated in conscious rabbits after oral administration together with intravenous doses of sodium nitroprusside (SNP). The effects of vardenafil were compared with those of the well-recognized PDE5 inhibitor, sildenafil (values for sildenafil in brackets). Vardenafil specifically inhibited the hydrolysis of cGMP by PDE5 with an IC50 of 0.7 nM (6.6 nM). In contrast, the IC50 of vardenafil for PDE1 was 180 nM; for PDE6, 11 nM; for PDE2, PDE3 and PDE4, more than 1000 nM. Relative to PDE5, the ratios of the IC50 for PDE1 were 257 (60), for PDE6 16 (7.4). Vardenafil significantly enhanced the SNP-induced relaxation of human trabecular smooth muscle at 3 nM (10 nM). Vardenafil also significantly potentiated both ACh-induced and transmural electrical stimulation-induced relaxation of trabecular smooth muscle. The minimum concentration of vardenafil that significantly potentiated SNP-induced cGMP accumulation was 3 nM (30 nM). In vivo studies in rabbits showed that orally administered vardenafil dose-dependently potentiated erectile responses to intravenously administered SNP. The minimal effective dose that significantly potentiated erection was 0.1 mg/kg (1 mg/kg). The selectivity for PDE5, the potentiation of NO-induced relaxation and cGMP accumulation in human trabecular smooth muscle and the ability to enhance NO-induced erection in vivo indicate that vardenafil has the appropriate properties to be a potential compound for the treatment of erectile dysfunction. Vardenafil was more potent and selective than sildenafil on its inhibitory activity on PDE5.