Presynaptic alpha 2-autoreceptors in rat and rabbit brain cortex were compared by means of antagonists and agonists. Brain cortex slices were preincubated with [3H]-noradrenaline and then superfused and stimulated by 3 (rat) or 4 (rabbit) pulses at a frequency of 100 Hz. The alpha 2-adrenoceptor agonist bromoxidine (UK 14304) reduced the electrically evoked overflow of tritium with EC50 values of 4.5 nmol/l in the rat and 0.7 nmol/l in the rabbit. The antagonists phentolamine, 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazo le (BRL 44408), rauwolscine, 1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo(c,f)imidazo(1,5-a)aze pine (BRL 41992), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4, 5-tetrahydro-3-benzazepine (SKF 104078), imiloxan, prazosin and corynanthine did not per se increase the evoked overflow of tritium but shifted the concentration-inhibition curve of bromoxidine to the right in a manner compatible with competitive antagonism. Up to 4 concentrations of each antagonist were used to determine its dissociation constant KD. The KD values correlated only weakly between the rat and the rabbit. Dissociation constants KA of bromoxidine were calculated from equieffective concentrations in unpretreated brain slices and slices in which part of the alpha 2-adrenoceptors had been irreversibly blocked by phenoxybenzamine. The KA value was 123 nmol/l in the rat and 7.2 nmol/l in the rabbit. The results confirm the species difference between rat and rabbit brain presynaptic alpha 2-autoreceptors. Comparison with data from the literature indicates that the rat brain autoreceptors can be equated with the alpha 2D subtype as defined by radioligand binding, whereas the rabbit brain autoreceptors conform to the alpha 2A subtype. For example, the antagonist affinities for the rat autoreceptors correlate with their binding affinities for the gene product of alpha 2-RG20, the putative rat alpha 2D-adrenoceptor gene (r = 0.97; P < 0.01), but not with their binding affinities for the gene product of alpha 2-C10, the putative human alpha 2A-adrenoceptor gene. Conversely, the rabbit autoreceptors correlate with the alpha 2-C10 (r = 0.98; P < 0.001) but not with the alpha 2-RG20 gene product. Since presynaptic alpha 2-autoreceptors are also alpha 2D in rat submaxillary gland and perhaps vas deferens and alpha 2A in rabbit pulmonary artery, the possibility arises that the majority of alpha 2-autoreceptors generally are alpha 2D in the rat and alpha 2A in the rabbit. Moreover, receptors of the alpha 2A/D group generally may be the main mammalian alpha 2-autoreceptors.