The binding characteristics of radiolabeled N6-(cyclohexyl)adenosine ([3H]CHA), N6-(R-phenylisopropyl)adenosine ([3H]R-PIA), 5'-N-ethyl-carboxamidoadenosine ([3H]NECA), and 2-[4-(2-carboxyethyl)phenyl]ethyl-amino-5'-N-ethylcarboxamidoadenosine ([3H]CGS 21680), to rat testis membranes were investigated. Specific binding of [3H]CGS 21680, a selective agonist for the A2a adenosine receptor, was very modest whilst the nonselective agonist [3H]NECA bound to rat testis membranes showing high binding capacity. At least two types of binding sites for [3H]NECA could be identified in rat testis membranes: high affinity sites and high capacity sites. Selective agonists for the A1 adenosine receptor, [3H]CHA and [3H]R-PIA bound with high affinity to a single class of binding sites. This high affinity binding site showed the typical pharmacological specificity of the A1 adenosine receptor with a potency order for agonists of CHA > or = R-PIA > NECA > N6-(S-phenylisopropyl)adenosine (S-PIA). In order to detect the presence of the A3 adenosine receptor in these membranes we selectively blocked the A1 receptor with a large molar excess of a xanthine antagonist, either 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) or xanthine amine congener (XAC). In the presence of an antagonist a low affinity binding site for [3H]CHA and [3H]R-PIA was detected. This low affinity binding site showed a different pharmacological specificity than the high affinity binding site. In fact the potency order for agonists was CHA > or = NECA = R-PIA > S-PIA. This finding suggests that the low affinity binding site represents the A3 adenosine receptor.