The antagonist radioligand 1,3-[3H]dipropyl-8-cyclopentylxanthine ([3H]DPCPX) was used to characterize adenosine A1 receptors in membrane preparations from atrial and ventricular myocardium of rat, rabbit, guinea pig and pig. Kd values in crude membranes from guinea pig atria and ventricles (3.3 and 3.0 nM) were higher than those in the other species (ranges, 1.5-1.8 and 1.5-1.9 nM). Bmax values were greater in atria than in ventricles in all four species, and in atria and ventricles of guinea pig (76 and 34 fmol/mg), than in the other species (ranges, 15-17 and undetectable to 12 fmol/mg). In contrast, guinea pig Kd and Bmax values for beta-adrenoceptors, which were labelled with (-)3-[125I]iodocyanopindolol, fell within the range of values for the other three mammalian species. In semipurified membrane preparations from pig, [3H]DPCPX and the agonist radioligand [125I]-N6-4-aminobenzyladenosine appeared to label a similar population of receptors and gave comparable Kd values in atria (0.73 and 0.66 nM) and in ventricles (0.57 and 0.70 nM). In semipurified preparations from pig, the agonist R-(-)-N6-(2-phenylisopropyl)adenosine (R-PIA) displaced [3H]DPCPX in a manner consistent with the presence of both high- and low-affinity adenosine A1 receptors. The data from this study indicate that the density of adenosine A1 receptors in atria is greater than in ventricles, but similar Kd values suggest that the A1 receptor population is the same in the two cardiac tissues. Also, the data demonstrate that the [3H]DPCPX antagonist binding characteristics of guinea pig myocardium differ from those in rat, rabbit and pig.