Presynaptic α2-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 α2-adrenoceptor agonist bromoxidine (UK 14 304) 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-dihydroimidazole (BRL 44408), rauwolscine, 1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo(c,f)imidazo(1,5-a)azepine (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 α2-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 α2-autoreceptors. Comparison with data from the literature indicates that the rat brain autoreceptors can be equated with the α2D subtype as defined by radioligand binding, whereas the rabbit brain autoreceptors conform to the α2A subtype. For example, the antagonist affinities for the rat autoreceptors correlate with their binding affinities for the gene product of α2-RG20, the putative rat α2D-adrenoceptor gene (r = 0.97; P<0.01), but not with their binding affinities for the gene product of α2-C10, the putative human α2A-adrenoceptor gene. Conversely, the rabbit autoreceptors correlate with the α2-C10 (r = 0.98; P<0.001) but not with the α2-RG20 gene product. Since presynaptic α2-autoreceptors are also α2D in rat submaxillary gland and perhaps vas deferens and α2A in rabbit pulmonary artery, the possibility arises that the majority of α2-autoreceptors generally are α2D in the rat and α2A in the rabbit. Moreover, receptors of the α2A/D group generally may be the main mammalian α2-autoreceptors.