Myenteric plexus-longitudinal muscle strips isolated from the small intestine of rats were incubated with [3H]choline to measure the synthesis and the release of [3H]acetylcholine. To separate different radioactive compounds (acetylcholine, choline, phosphorylcholine) from both the tissue and the overflow a new method, the reverse phase HPLC, was used. The radiochromatogram following the injection of a [3H]choline-standard and a [14C]acetylcholine-standard onto the HPLC showed a clear separation of both isotopes with a recovery rate of roughly 100%. Incubation of the muscle strips with [3H]choline caused the synthesis of [3H]acetylcholine (30,000 dpm/preparation) that increased 2-fold, when the electrical field stimulation during labelling was increased from 0.2 Hz to 1 Hz. Electrical field stimulation (3 Hz, 2 min) caused an increase in tritium efflux that was abolished by the removal of extracellular calcium or by the addition of tetrodotoxin. Analysis by reverse phase HPLC of the overflow showed that the stimulated increase in tritium overflow was balanced by the enhanced release of [3H]acetylcholine. whereas the overflow of [3H]choline was not affected by the electrical field stimulation. Oxotremorine (1 mumol/l) suppressed the release of [3H]acetylcholine by 60%. Scopolamine (0.1 mumol/l) prevented this inhibition and, given alone, enhanced the release of [3H]acetylcholine by 43%. The release of [3H]acetylcholine evoked at 0.2, 2 or 20 Hz did not consistently decline at increasing frequencies. The present experiments show the synthesis and the calcium-dependent release of [3H]acetylcholine from the myenteric plexus-longitudinal muscle preparation of rats correspondingly to the same in-vitro preparation isolated from guinea-pigs. Muscarinic autoinhibition operates also in the small intestine of rats.(ABSTRACT TRUNCATED AT 250 WORDS)