The effect of pertussis toxin pretreatment on the putative A3 receptor-mediated hypotensive response to N6-2-(4-aminophenyl)ethyladenosine (APNEA) has been investigated in the anaesthetised rat. Pertussis toxin pretreatment essentially abolished the bradycardia induced by the prototype A1 receptor agonist, N6-cyclopentyladenosine, whereas the fall in blood pressure induced by the selective A2A receptor agonist, CGS 21680 was enhanced. Pertussis toxin substantially reduced the hypotensive response to APNEA. In this respect, the mechanism of action resembles that of the cloned A3 receptor which when expressed in CHO cells couples negatively to adenylate cyclase by a pertussis toxin-sensitive mechanism. The data provide further evidence that adenosine A3 receptors mediate the hypotensive response to APNEA in the rat.