Penile erection is mediated by nitric oxide (NO) synthesized by the neuronal nitric oxide synthase (nNOS). In the rat penis, the main nNOS mRNA variant, PnNOS, differs from cerebellar nNOS (CnNOS) by a 102 base pair insert encoding a 34-amino acid sequence. In the mouse, two nNOS mRNAs have been identified: nNOSα, encoding a 155-kDa protein, and an exon 2-deletion variant, nNOSß, encoding a 135-kDa protein that lacks a domain where a protein inhibitor of nNOS (PIN) binds. We wished to determine whether PnNOSα and β are expressed in the rat penis and are located in the nerves and whether the β form persists in the potent nNOS knock-out mouse (nNOS^△△). A PnNOS antibody against the insert common to both PnNOSα and β detected the expected 155-kDa protein in PnNOSα-transfected cells. This antibody, and the one common to PnNOS/CnNOS, showed (on Western blots) the 155- and 135-kDa nNOS variants in rat penile tissue during development and aging. PnNOSα mRNA and its subvariants were found as the main nNOS in the penile corpora, the cavernosal nerve, and the pelvic ganglia, with lower levels of PnNOSβ mRNA. In tissue sections, PnNOS protein was immunodetected in the penile nerve endings in the rat and in the nNOS wild-type and nNOS^△△ mice. An antibody against the sequence encoded by exon 2 did not react (on Western blots) with the 135-kDa band, which confirms that this protein is the β form. In conclusion, both PnNOSα and β are expressed in the rat penis at all ages and are located in the nerves. The β form may allow nitric oxide synthesis during erection to be partially insensitive to PIN. The residual expression of PnNOS, and possibly CnNOS, in the penis of the nNOS^△△ mouse occurs through transcription of the β mRNA, and this may explain the retention of erectile function when the expression of nNOSα is disrupted.