Contractility of the peritubular smooth muscle layer ensures the transit of immotile spermatozoa through the epididymal duct to acquire their fertilizing capacity. Atrial natriuretic peptide (ANP) and nitric oxide (NO) affect contractility via cGMP signals that are controlled by phosphodiesterases (PDEs). Sildenafil inhibits the cGMP-hydrolyzing PDE5 and thereby promotes relaxation of smooth muscle cells. While sildenafil is increasingly used in young patients for the treatment of pulmonary hypertension, virtually no knowledge exists about PDEs in the epididymis. Western blotting, immunohistochemistry and RT-PCR analyses after laser capture microdissection localized PDE5 to smooth muscle cells, but not to epithelial cells, of the epididymal duct in man and rat. Sildenafil, ANP and NO significantly slowed spontaneous contractions of rat epididymal duct segments in organ bath studies. Sildenafil effects were additive to ANP and NO. Long-term exposure to sildenafil in vivo did not change the PDE5 expression or the observed contractility pattern with the rapid relaxing response toward ANP, NO and sildenafil. Data demonstrate that PDE5 is an important member of cGMP signaling pathways regulating the finely orchestrated process of epididymal duct contractility and suggest, however, that in the epididymis side effects of therapeutically used sildenafil are unlikely.