We recently reported the cloning of a cDNA (designated RFL9) that encodes a novel A2-adenosine receptor subtype. We now fully characterize the pharmacological properties of RFL9 in stably transfected CHO cells by examining cAMP responses to drug treatments. The pharmacological profile of cAMP responses in RFL9-transfected cells was similar to that expected for A2b-adenosine receptors and distinct from that of CHO cells transfected with the A2a-adenosine receptor. When RFL9-transfected cells were compared with VA 13 fibroblasts, the human cell line in which endogenous A2b-adenosine receptors were originally characterized, the dose-response curves of cAMP responses to drug treatments were highly correlated. Northern blot analysis of RNA prepared from VA 13 fibroblasts revealed specific hybridizing transcripts when probed for RFL9, but no hybridizing signal for A2a-adenosine receptor mRNA. Using degenerate oligonucleotide primers designed to detect adenosine receptors by the polymerase chain reaction, only one cDNA fragment homologous to the rat A2b-adenosine receptor was isolated from VA 13 cells. These results strongly suggest that RFL9 encodes the proposed A2b-adenosine receptor subtype. The identification of the cDNA for an A2b-adenosine receptor will allow more rigorous characterization of its anatomical distribution and functional properties.