Rationale: Sildenafil has been implicated in the activation of cystic fibrosis transmembrane conductance regulator (CFTR) protein. The effect was observed in vitro and in the presence of doses roughly 300 larger than those commonly used for treating erectile dysfunction. Objectives: To evaluate in vivo the therapeutic efficacy of clinical doses of sildenafil and vardenafil, two clinically approved phosphodiesterase 5 inhibitors, for activating ion transport in cystic fibrosis. Methods: We measured transepithelial potential difference in vivo across the nasal mucosa as a readout for sodium and chloride transport. The effect of a single intraperitoneal injection of sildenafil (0.7 mg/kg) or vardenafil (0.14 mg/kg) was investigated in F508del, cftr knockout and normal homozygous mice. Measurements and Main Results: In F508del mice, but not in cftr knockout mice, the chloride conductance, evaluated by perfusing the nasal mucosa with a chloride-free solution in the presence of amiloride and with forskolin, was corrected 1 h after sildenafil administration. A more prolonged effect, persisting for at least 24 h, was observed with vardenafil. The forskolin response was increased after sildenafil and vardenafil in both normal and F508del mutant animals. In F508del mice, the chloride conductance in the presence of 200 µM 4-4' diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of alternative chloride channels, was much higher after sildenafil injection than following placebo treatment. No effect on the sodium conductance was detected in any group of animals. Conclusions: Our results provide preclinical evidence that both drugs stimulate chloride transport activity of F508del-CFTR protein.