Longitudinal muscle strips adhered with myenteric plexus were subjected to enzyme digestion under controlled conditions in a Krebs-bicarbonate buffer solution containing a mixture of collagenase, deoxyribonuclease, protease, choline chloride, and bovine serum albumin for 30 min at 37 degrees C. Myenteric ganglia, singly or in multiple aggregates, were harvested with micropipette and labeled with [3H]choline for [3H]acetylcholine (ACh) release studies. When examined by light or electron (transmission or scanning) microscopy, the ganglia exhibited their normal structural characteristics with axon bundles, dendrites, cell bodies, and vesiculated processes. Depolarization with elevated potassium or veratrine hydrochloride significantly elevated the efflux of [3H] ACh. Perfusion with tachykinins (substance P and substance K), vasoactive intestinal peptide, forskolin, or serotonin also significantly increased the release of [3H]ACh. This study demonstrated that enzyme-dissociated myenteric ganglia, notably free of muscle or connective tissue components, were structurally well preserved and were amenable to functional studies targeted specifically for the enteric plexus neurons.