Binding of the alpha 2-adrenoceptor antagonist [3H]-rauwolscine was characterized in membrane preparations from the kidneys of mouse, rat, rabbit, dog, and man. In all species, binding reached equilibrium within 45 min and dissociated at a single exponential rate after addition of phentolamine 10 microM. Saturation studies showed that the affinity of [3H]-rauwolscine was similar in all species (2.33-3.03 nM) except man where it was significantly higher (0.98 nM). Marked differences were seen in the density of binding sites, increasing in the order: man less than dog less than rabbit less than rat less than mouse. In all cases, Hill coefficients were not significantly different from unity. [3H]-rauwolscine binds with low affinity (KD greater than 15 nM) to membranes prepared from guinea-pig kidney. The low affinity binding is not due to the absence of particular ions in the incubation medium or to receptor occupation by endogenous agonist. The binding in all species was found to be stereoselective with respect to the isomers of noradrenaline. However, differences were seen in the characteristics of agonist interactions with the binding site both between isomers and between species. Marked differences in affinity of particular alpha-adrenoceptor antagonists were observed for alpha 2-adrenoceptors labelled by [3H]-rauwolscine. These differences were most evident with the alpha 1-adrenoceptor selective antagonist prazosin which displayed inhibition constants (Ki values) of 33.2, 39.5, 261, 570 and 595 nM in rat, mouse, dog, man and rabbit, respectively. Differences are apparent in the characteristics of alpha 2-adrenoceptors labelled by [3H]-rauwolscine between species and it is suggested that the differences observed for alpha 1-selective antagonists such as prazosin may be related to binding to additional sites in the vicinity of the alpha 2-adrenoceptor.