Introduction Recent electrophysiological recordings have identified the existence of outward, excitatory chloride (Cl−) currents in rat, rabbit, and human corpus cavernosum (CC) muscle cells. Aim The aim of this study was to investigate the physiological role of Cl− currents in the maintenance of cavernosal muscle tone in isolated rabbit CC tissues. Methods CC strips (1 × 1 × 5 mm) were suspended in tissue bath chambers for isometric tension experiments. Spontaneous cavernosal tone and contractions elicited by field stimulation or administration of established smooth muscle constrictors were examined in the presence of chloride channel (ClC) blockers, ni umic acid (NFA), and anthracene-9-carboxylic acid (A9C). Main Outcome Measure Both spontaneous myogenic activity and contractile responses to field stimulation, norepinephrine, histamine, and endothelin-1 were reduced by ClC blockers. Results In CC strips exhibiting intrinsic myogenic tone, NFA (30 and 100 µM) and A9C (1 mM) caused a relaxation of the tone. In addition, spontaneous contractile activity in CC was abolished in the presence of either ClC blocker. In CC strips precontracted with norepinephrine, histamine, and endothelin-1, both ClC blockers significantly reversed the tone. The ability of NFA and A9C to reverse norepinephrine-induced tone was unaffected by N  ω-nitro-L-arginine, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, and cis-N-[2-phenylcyclopentyl]-azacyclotridec-1-en-2-amine. In addition, neither indomethacin nor tetraethylammonium affected the relaxant response of NFA. NFA and A9C inhibited the neurogenic and norepinephrine-induced contractions in a concentration-dependent manner. While NFA exerted persistent inhibition on neurogenic contraction, inhibition of neurogenic contractions by A9C was readily reversible. On K+-depolarized CC, NFA induced a concentration-related relaxation, whereas A9C was inert, suggesting an additional mechanism of NFA on voltage-gated calcium channels. Conclusions These results underline the importance of Cl− currents as a mechanism in the maintenance of cavernosal tone produced by adrenergic and various endogenous constrictors. Thus, the modulation of Cl− current, as an attractive and effective approach to regulate penile erection, and specific ClC blockers, as potential erectogenic agents, merits further research.