Radioligand binding to A1 adenosine receptors at brain membranes from seven species was investigated. The antagonist 8-cyclopentyl-1,3-[3H]dipropylxanthine ([3H]DPCPX) bound with affinities between 0.17 nM in sheep brain and 2.1 nM in guinea pig brain. Competition of several antagonists for [3H]DPCPX binding showed that the most potent compounds were DPCPX with Ki values of 0.05 nM in bovine brain and 1.1 nM in guinea pig brain and xanthine amine congener (XAC) with Ki values of 0.03 nM in bovine brain and 5.5 nM in guinea pig brain. The differences in affinity of the agonist radioligand 2-chloro-N6-[3H]cyclopentyl-adenosine ([3H]CCPA) were less pronounced, ranging from a KD value of 0.12 nM (hamster brain) to 0.42 nM (guinea pig brain). Agonist competition for [3H]DPCPX binding of photoaffinity labelling, however, exhibited marked species differences. N-Ethylcarboxamidoadenosine (NECA) and S-N6-phenylisopropyladenosine (S-PIA) showed 20 to 25-fold different KD values in different species. NECA had a particularly high affinity in guinea pig brain and was only two-fold less potent than R-PIA. Thus, the difference from the "classical" A1 receptor profile (R-PIA greater than -NECA greater than S-PIA) is not sufficient to speculate that A1 receptor subtypes may exist that are coupled to different effector systems. Our data show that these difference can easily be explained by species differences.