The study was designed to classify in terms of alpha 2A, alpha 2B, alpha 2C and alpha 2D the presynaptic alpha 2-autoreceptors, as well as the alpha 2-receptors modulating the release of acetylcholine, in the myenteric plexus-longitudinal muscle (MPLM) preparation of the guinea-pig ileum. A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the MPLM preparation were preincubated with 3H-noradrenaline or 3H-choline and then superfused and stimulated electrically. The stimulation periods used (3H-noradrenaline: 3 trains of 20 pulses, 50 Hz, train interval 60 s; 3H-choline: single trains of 30 pulses, 0.2 Hz) did not lead to alpha 2-autoinhibition or inhibition of 3H-acetylcholine release by endogenous noradrenaline. The alpha 2-selective agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) reduced the evoked overflow of tritium in both 3H-noradrenaline and 3H-choline experiments. Most (3H-noradrenaline) or all (3H-choline) of the 10 antagonists shifted the concentration-inhibition curves of UK 14,304 to the right. pKd values of the antagonists were calculated from the shifts. pKd values from 3H-noradrenaline experiments correlated with pKd values from 3H-choline experiments (r = 0.981). It is concluded that alpha 2-autoreceptors and alpha 2-heteroreceptors modulating the release of acetylcholine in the MPLM preparation are of the same subtype. Comparison with antagonist affinities for prototypic native alpha 2 binding sites, binding sites in cells transfected with alpha 2 subtype genes, and previously classified presynaptic alpha 2-adrenoceptors--all taken from the literature--indicates that both are alpha 2D.(ABSTRACT TRUNCATED AT 250 WORDS)