Sildenafil and tadalafil are widely-used phosphodiesterase 5 (PDE5) inhibitors for which no clear dose-response relationship could be established. Using isolated and/or recombinant PDE5, it has been demonstrated that cGMP can increase the affinity of this enzyme for sildenafil and tadalafil. We thus hypothesized that in cells expressing the nitric oxide – soluble guanylyl cyclase – cyclic guanosine monophosphate – PDE5 (NO-sGC-cGMP-PDE5) pathway such as platelets, the presence of NO increases the intracellular cGMP content and thus promotes the intracellular accumulation of sildenafil or tadalafil. As a cell model, isolated and washed human platelets were used. Platelet suspensions were incubated with sildenafil or tadalafil at different concentrations and for various time intervals with or without an NO donor to increase intraplatelet cGMP concentrations. Intracellular sildenafil or tadalafil was quantified by ultra-performance liquid chromatography tandem mass spectrometry and intracellular cGMP by an enzyme-linked immunosorbent assay. Sildenafil accumulated in platelets with an up to 4-fold higher accumulation when platelets were pretreated with an NO donor (p<.0001). Accumulation of tadalafil in platelets was even higher, whereas the increase was 2-fold when an NO donor was present (p<.001). This accumulation was time-dependent and happened concomitantly with a rise in intracellular cGMP. Our data demonstrate that intracellular cGMP increases intracellular PDE5 inhibitor concentrations most likely by raising the affinity of these compounds for PDE5. These findings suggest that PDE5 inhibitor action in humans is critically influenced by modulators of the activity of the NO pathway.