NADPH-diaphorase histochemistry was combined with demonstration of acetylcholinesterase and immunocytochemistry for calcitonin gene-related peptide to study esophageal innervation in the rat. Most of the myenteric neurons stained positively for NADPH-diaphorase, as did numerous varicose nerve fibers in the myenteric plexus, among striated muscle fibers, around arterial blood vessels, and in the muscularis mucosae. A majority of motor endplates (as demonstrated by acetylcholinesterase histochemistry or calcitonin gene-related peptide immunocytochemistry) were associated with fine varicose NADPH-diaphorase-positive nerve fibers. Analysis of brainstem nuclei, sensory vagal, spinal, and sympathetic ganglia in normal and neonatally capsaicin-treated rats, and comparison with anterogradely labeled vagal branchiomotor, preganglionic and sensory fibers led to the conclusion that NADPH-diaphorase-positive fibers on motor endplates originate in esophageal myenteric neurons. No association of NADPH-diaphorasepositive nerve fibers with motor endplates was found in other organs containing striated muscle. These results suggest extensive, presumably nitrergic, co-innervation of esophageal striated muscle fibers by enteric neurons. Thus, control of peristalsis in the esophagus of the rat may be more complex than hitherto assumed.