1. We characterized the binding of [3H]-rauwolscine, [3H]-p-aminoclonidine and [3H]-idazoxan in a dog kidney membrane preparation. Our aim was to determine the pharmacological nature of the alpha 2-adrenoceptor- and imidazoline-preferring binding sites in this organ. 2. [3H]-Rauwolscine bound to an apparent single site with an affinity (KD) of 2.2 nmol/L and a maximum density (Bmax) of 58.5 fmol/mg protein, when 10 mumol/L idazoxan defined non-specific binding. However displacement studies demonstrated that a number of compounds, including prazosin, inhibited [3H]-rauwolscine binding in a complex manner consistent with displacement from two distinct binding sites. The majority (69%) of the [3H]-rauwolscine binding sites had a relatively low affinity for prazosin (KI = 398 nmol/L), while the remainder had a relatively high affinity for prazosin (KI = 7.9 nmol/L). 3. [3H]-p-Aminoclonidine bound to an apparent single site (KD = 5.2 nmol/L; Bmax = 72.4 fmol/mg protein), when 10 mumol/L phentolamine defined non-specific binding. When 1 mumol/L of the potent and selective alpha 2-adrenoceptor antagonist 2-methoxyidazoxan was included in the incubate, no specific binding was detected. We therefore conclude that under the conditions of this experiment [3H]-p-aminoclonidine binds only to alpha 2-adrenoceptors in the dog kidney. 4. [3H]-Idazoxan bound to two sites, with a higher (KD = 0.95 nmol/L; Bmax = 43.9 fmol/mg protein) and lower (KD = 9.1 nmol/L; Bmax = 93.8 fmol/mg protein) affinity, respectively, when 1 mmol/L phentolamine defined non-specific binding. When 10 mumol/L GTP gamma S was included in the incubate, the low affinity site was unaffected but the maximum binding at the higher affinity site was reduced by 79%. 2-Methoxyidazoxan displaced [3H]-idazoxan in a monophasic manner and with low potency (IC50 = 11.5 mumol/L). Yohimbine, efaroxan, clonidine, rilmenidine, guanabenz and idazoxan itself displaced [3H]-idazoxan in a complex manner; the slope of the displacement curves being less than unity. 5. We conclude that the dog kidney contains a heterogeneous population of alpha 2-adrenoceptors that can be labelled either with [3H]-rauwolscine or [3H]-p-aminoclonidine. The dog kidney also contains a heterogeneous population of non-adrenoceptor imidazoline-preferring binding sites of the I2-subtype, that can be labelled with [3H]-idazoxan. The binding site for which [3H]-idazoxan has the highest affinity appears to be coupled to a guanine nucleotide binding regulatory protein.