The role of adenosine A3 receptors on human eosinophil degranulation and superoxide anion (O2-) release was studied in vitro using the complement fragment C5a as the main stimulus and employing a number of selective agonists and antagonists. In the presence of cytochalasin B (CB), C5a induced a dose-dependent release of the granular eosinophil peroxidase (EPO), but not O2-, whereas in the absence of CB O2- , but not EPO, was released. C5a-induced EPO release was inhibited dose-dependently by the selective A3 agonist N6-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine (IB-MECA) and to a lesser extent by the less-selective N6-2-(4-amino-3-iodophenyl) ethyladenosine (APNEA). The IC50 (95% CI) for IB-MECA was 6.8 microM (3.1-12.0 microM). At concentrations up to 100 microM, neither adenosine nor the selective A1 agonist N-cyclopentyladenosine (CPA) and the selective A2 agonist 2-[[2-[4-(2-carboxyethyl)phenyl]ethyl]amino]-N-ethylcarboxamidoadenosine (CGS 21680) had any significant effect. The inhibitory effect of IB-MECA was almost completely abolished by pre-treatment with 1 microM of the selective A3 antagonist 9-chloro-2-(2-furyl)-5-phenylactylamino[1,2,4]triazolo[1,5-c]quina zoline (MRS 1220), but not the selective A1 antagonist 1,3-dipropyly-8-cyclopentylxanthine (DPCPX) or the selective A2 antagonist 3,7-dimethyl-1-propargylxanthine (DMPX). IB-MECA also significantly inhibited C5a-induced O2- release with IC50 (95% CI) of 9.5 microM (4.6-13.1 microM) whereas adenosine and the A1 agonist CPA potentiated this effect at low concentrations. The potentiation appeared to be a result of their direct O2- release from these cells, probably mediated via A1 receptors. The inhibition by IB-MECA was selectively reversed by MRS 1220. These results show that the A3 receptors on human eosinophils mediate inhibition of both degranulation and O2- release and suggest a therapeutic potential for A3 agonists in diseases such as asthma in which activated eosinophils are involved.