Background: The N-methyl-D-aspartate (NMDA)/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway has been implicated in the neurobiology of depression. Recently we suggested a possible complex interaction between the cholinergic and NO-cGMP pathways in the antidepressant-like response of the phosphodiesterase type 5 (PDE5) inhibitor, sildenafil. Specifically, we demonstrated that chronic (11 days) treatment with a combination of sildenafil (10 mg/kg/day) + atropine (1 mg/kg/day) produces antidepressant-like effects in the forced swim test (FST) in Sprague Dawley rats. Neither of these drugs produced any antidepressant-like effect when administered alone. In the current study, we investigated these findings in a genetic animal model of depression, the Flinders Sensitive Line (FSL) rats. In addition, we evaluated the dose-dependency and onset of action for sildenafil + atropine, as well as the efficacy of various augmentation strategies in combination with fluoxetine and imipramine. Methods: Treatment regimes: FSL rats were injected intraperitoneally daily with sildenafil (0, 1, 3, 10 and 20 mg/kg/day) ± atropine (1 mg/kg/day), fluoxetine (5 mg/kg/day) or imipramine (15 mg/kg/day) for 14 days. Rats were also treated with sildenafil (10 mg/kg/day) ± atropine in combination with fluoxetine (5 mg/kg/day) or imipramine (15 mg/kg/day) for 7 and 14 days. Behavioural testing: On the last day of treatment, approximately 5 hours into the dark-cycle (±12 hours following the last injection) immobility was scored during five minutes swim in the FST. In addition, locomotor activity was evaluated in the Open Field Test 2 hours prior to the FST. Results: Fluoxetine and imipramine separately decreased immobility in FSL rats, comparable to that of FRL control rats, after 14 but not after 7 days. Likewise, when combined with atropine, sildenafil also decreased immobility in FSL rats at doses of 3, 10 and 20 mg/kg/day with a dose-dependent trend, but not with 1 mg/kg/day. While fluoxetine + sildenafil + atropine was not superior to fluoxetine alone, there was a trend for augmentation of imipramine when combined with sildenafil after 14 days. Importantly, only sildenafil + imipramine in combination produced a significant antidepressant-like effect in FSL rats after 7 days. Locomotor activity was unaltered by all treatments, indicating that reduced immobility in the FST was not secondary to increased locomotor activity. Conclusions: Using a genetic animal model of depression, we have confirmed the antidepressant-like property of sildenafil following “unmasking” by concomitant block of muscarinic receptors. These findings hint at a novel interaction between the cGMP and cholinergic systems in depression, and suggest a strategy for the treatment of depression, using a PDE5 inhibitor in the presence of cholinergic inhibition. Sildenafil-induced augmentation of imipramine, an antidepressant with inherent anticholinergic properties, concurs with this suggestion, and highlights the potential clinical value of this observation.