South American (SA) opossum lower esophageal sphincter (LES) circular smooth muscle relaxes by activation of enteric nerves elicited by EFS (electrical field stimulation, 0.5 ms, 48 V, 0.5–8 Hz for 10 s). The identity of the mediator released and the cellular mechanism, however, remain to be fully elucidated. The purpose of this study was to determine the effect of the enzyme soluble guanylate cyclase (cGC) inhibitors, cystamine (100 μM), methylene blue (30 μM), LY 83583 (6-anilino-5,8 quinoledione, 10 μM) and ODQ (H-[1,2,4]oxadiazolo[4,3]quinoxalin-1-one, 1 μM) on the relaxations induced by EFS and by exogenous NO (nitric oxide, 0.5 mM) or NO-donors on SA opossum LES smooth muscle strips. EFS caused frequency-dependent relaxations, which were inhibited by NO-synthase inhibitors and abolished by tetrodotoxin. Cystamine did not affect relaxations caused by EFS and NO or NO-donor. Methylene blue also failed to affect EFS-caused relaxations, although it was capable of inhibiting relaxation induced by NO. LY 83583 inhibited relaxations induced by NO, but did not affect those induced by EFS or by SNAP and HXA. ODQ abolished relaxations caused by EFS at lower frequencies and by HXA (hydroxylamine, 10 μM) and SNAP (S-nitroso-N-acetyl penicillamine, 10 μM). Relaxations at higher frequencies of EFS and induced by SNP (sodium nitroprusside, 30 μM) and NO were only reduced by ODQ. These findings indicate that activation of the cGC can be involved in relaxations induced by EFS at lower frequencies, but other mechanisms can be involved at higher frequencies of EFS and caused by SNP or NO.