The study was devised to classify, by means of antagonist affinities, the presynaptic alpha 2-autoreceptors of rabbit atria and kidney in terms of alpha 2A, alpha 2B, alpha 2C and alpha 2D. A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the left atrium and slices of the kidney cortex were preincubated with 3H-noradrenaline and then superfused and stimulated electrically. In one series of experiments, tissue pieces were stimulated by relatively long pulse trains (1 or 2 min) leading to alpha 2-autoinhibition. All 11 (atria) or 10 (kidney) antagonists increased the evoked overflow of tritium. pEC30% values (concentrations causing 30% increase) were interpolated from concentration-response curves. In a second series of experiments, tissue pieces were stimulated by brief pulse trains (0.4 s) that did not lead to alpha 2-autoinhibition, and concentration-inhibition curves of the alpha 2-selective agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) were determined. Most of the 11 (atria) or 8 (kidney) antagonists shifted the concentration-inhibition curve of UK 14,304 to the right. pKd values of the antagonists were calculated from the shifts. pEC30% values correlated with pKd values, both in atria (r = 0.728) and in the kidney (r = 0.930). pEC30% values in atria correlated with pEC30% values in the kidney (r = 0.988) and pKd values in atria correlated with pKd values in kidney (r = 0.923). It is concluded that the alpha 2-autoreceptors in atria and the kidney are the same. Comparison with antagonist affinities for prototypic native alpha 2 binding sites, alpha 2 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 autoreceptors are alpha 2A. This conclusion is reached with either of the two independent estimates of autoreceptor affinity, pEC30% and pKd. The results are compatible with the hypothesis that at least the majority of alpha 2-autoreceptors belong to the alpha 2A/D branch of the alpha 2-adrenoceptor tree, across mammalian or at least rodent and lagomorph species.