The molecular mechanism for priapism is not well characterized. Although the nitric oxide (NO) pathway is known to mediate penile erection under normal conditions, we hypothesized that the mechanism of priapism rests in aberrant downstream signaling of this pathway based on our previous findings that mice lacking the gene for endothelial nitric oxide synthase (eNOS^—/—) and mice lacking both neuronal NOS (nNOS) and eNOS (nNOS^—/—, eNOS^—/—) have a tendency for priapic activity. We investigated the role of downstream guanylate cyclase and phosphodiesterase type 5 (PDE5A) expression and function in mediating these responses in eNOS^—/— and nNOS^—/—, eNOS^—/— mice. Erectile responses to both cavernous nerve stimulation and intracavernosal injection of the NO donor diethylamine-NONOate were augmented in eNOS^—/— and nNOS^—/—, eNOS^—/— mice but not in WT or nNOS^—/— mice. PDE5A protein expression and activity and cGMP levels were significantly lower in eNOS^—/— and nNOS^—/—, eNOS^—/— mice, and this effect was reproduced in WT corpus cavernosum exposed to NOS inhibitors. Moreover, cavernous nerve stimulation was associated with a marked augmentation of cavernosal cGMP levels, suggesting that, although lower at baseline, the production of cGMP is unchecked in eNOS^—/— and nNOS^—/—, eNOS^—/— mice upon neurostimulation. Transfection of eNOS^—/— mice with an adenovirus encoding eNOS resulted in a normalization of PDE5A protein and activity as well as a correction of priapic activity. Coupled with the observation that sickle cell disease mice (which show a priapism phenotype) evince dysregulated PDE5A expression/activity, these data suggest that PDE5A dysregulation is a fundamental mechanism for priapism.