The lower esophageal sphincter (LES) has a circular muscle component exhibiting spontaneous tone that is relaxed by nitric oxide (NO), and a low tone sling muscle that contracts vigorously to cholinergic stimulation, but with little or no evidence of NO responsiveness. This study dissected the responses of the sling muscle to nitrergic innervation in relationship to its cholinergic innervation and the circular muscle responses. Motor responses were induced by electrical field stimulation (EFS, 1-30 Hz) of muscle strips from sling and circular regions of the feline LES, in presence of cholinergic receptor inhibition (atropine) or NO synthase inhibition (L-NNA ±atropine). This study shows 1) Sling muscle developed less intrinsic resting tone compared to circular muscle. 2) With EFS, a) sling muscle contracted, most at 10 Hz, whereas b) circular muscle relaxed >50% by 5 Hz. 3) On neural blockade with atropine or L-NNA ±atropine: a) Sling muscle, although predominantly influenced by excitatory cholinergic stimulation, had a small neural NO-mediated inhibition, with no significant non-NO-mediated inhibition; b) Circular muscle, although little affected by cholinergic influence, underwent relaxation predominantly by neural release of NO and some non-NO inhibitory influence (at higher EFS frequency). 4) The sling, pre-contracted with bethanecol, could relax with NO and some non-NO inhibition. 5) The tension range of both muscles is similar. In conclusion, sling muscle has limited NO-mediated inhibition to potentially augment or replace sling relaxation effected by switching off its cholinergic excitation. Differences within the LES sling and circular muscles could provide new directions for therapy of LES disorders.