To examine possible species differences in pharmacology, rat adenosine A2A receptors were studied in PC12 (pheochromocytoma) cells, and human receptors in Chinese hamster ovary (CHO) cells transfected with the cloned human A2A receptor cDNA. Using [3H]-5-amino-7(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine ([3H]-SCH 58261) as radioligand, the estimated Bmax (maximal binding) was 538 and 2085 fmol/mg in CHO and PC12 cells, respectively. The Kd (dissociation constant) values for [3H]-SCH 58261 were 1.05 and 5.6 nM in the two cell types, respectively. The order of potency of antagonists and most agonists was the same in both cell types, but 2-phenylaminoadenosine and 2-chloroadenosine were relatively less potent in PC12 cells than in CHO cells. In the functional assay, using cyclic AMP accumulation, all agonists tested were more potent in CHO than in PC12 cells, but this could not be readily explained by differences in adenylyl cyclase or in the expression of G proteins. As in the case of binding, the relative agonist potencies were similar for most compounds, but 2-phenylaminoadenosine and 2-chloroadenosine were more potent at human A2A receptors in CHO cells than predicted from the data obtained on rat A2A receptors in PC12 cells. Antagonists were approximately equipotent in the two cells. These results show that, despite only small differences in amino acid sequences and no difference in antagonist pharmacology, the relative order of potency of receptor agonists can differ between species homologues of the adenosine A2A receptor.