The aberrant functioning of the A1 adenosine receptor of adipose tissue has been implicated as a factor in obesity. To begin to address questions concerning this relationship, the possibility of a unique A1 adenosine receptor in adipose tissue must be investigated. Therefore, cDNAs encoding the A1 adenosine receptors of adipose tissues of a mouse and an obese human were isolated, sequenced, and expressed in eukaryotic cells. We found their sequences to be 90% identical and each identical to published sequences of the receptors in brain preparations of the two species. The two cDNAs were transiently expressed in 293T cells, a human kidney cell line. Despite the 90% identity in their sequences, the ligand binding properties of the human and mouse cDNAs expressed in the 293T cell line differed markedly. With respect to amino acid differences in the extracellular loops, four occur in the second extracellular loop, which has been implicated in binding by other studies. The ligand binding characteristics of the recombinant receptors matched those of native receptors from human and mouse adipose tissue. The human A1 receptor cDNA was also expressed in ob17 preadipocyte cells to investigate reported influences of cellular environment on binding characteristics. We compared ligand binding of the expressed receptor in the two cell lines (ob17 and 293T). We also compared ligand binding of native receptors from mouse brain and adipose tissue preparations. In both studies, cellular environment had no affect on binding characteristics. This conclusive evidence resolves earlier conflicting reports in the literature.