The aim of this study was to characterize the kinetics of metabolite formation of the phosphodiesterase type-5 (PDE5) inhibitors sildenafil and tadalafil by CYP3A4, CYP3A5, and CYP3A7 isoforms. The formations of N-desmethyl sildenafil and desmethylene tadalafil were examined using CYP3A supersomes co-expressing human P450 oxidoreductase and cytochrome b 5 . Both sildenafil N-demethylation and tadalafil demethylenation were catalyzed by CYP3A4, CYP3A5, and to a lesser extent by CYP3A7. The kinetics of desalkyl metabolite formation of the two drugs were well fitted to the Hill equation; however, the Hill coefficients (n) suggested CYP3A-mediated negative cooperativity. Next, we analyzed the kinetics with a two binding sites model assuming two reaction steps: reaction 1 with high-affinity and low-capacity metabolism and reaction 2 with low-affinity and high-capacity metabolism. The kinetics of desalkyl metabolite formation were also fitted to the two binding sites model 1) PDE5 inhibitors have been shown to be an effective vasodilator, and have been used widely as a treatment not only for erectile dysfunction, but also for pulmonary arterial hypertension (PAH).2) Sildenafil and tadalafil are orally active inhibitors of PDE5 that are approved for the treatment of PAH in Japan, but their half-life period in the body differs substantially.3,4) That is, sildenafil has a fairly short half-life of about 4-5 h, whereas tadalafil has a long half-life of about 17.5 h in healthy subjects.5,6) The short half-life of sildenafil makes it the drug of choice in patients with more severe cardiovascular disease, allowing early use of supportive treatment if an adverse clinical event occurs. 7) In contrast, tadalafil has the advantage of once a day dosing compared to sildenafil's three times a day dosing, with implications for patient convenience and compliance. 6,8) Therefore, a transition from sildenafil to tadalafil has been frequently tried in stable patients with PAH. 3,8) In humans, sildenafil is eliminated predominantly by hepatic metabolism and is converted to an active metabolite, N-desmethyl sildenafil, with properties similar to the parent drug.9) The N-demethylation by CYP3A is the major route of metabolism of sildenafil in humans 10) (Fig. 1). Recently, Ku et al. 11) showed that both CYP3A4 and CYP3A5 played a significant role in the metabolism of sildenafil using CYP3A supersomes. That is, the intrinsic clearance for N-dealkylation of sildenafil by CYP3A5 and CYP3A4 were 0.09 and 0.07 µL/ min/pmol P450, respectively. 11) They also demonstrated that the mean rate for N-desalkyl metabolite formation from sildenafil was high in human liver microsome preparations with CYP3A5 activity (heterozygous for CYP3A5*1/*3 alleles) compared to those with null CYP3A5 activity (homozygous for CYP3A5*3 allele).11) These results suggested that genetic polymorphism of CYP3A5 at least partly contributes to interindividual variability in the disposition of sildenafil. On the other hand, tadalafil is eliminated predominantly by oxidative me...