BAY60-6583 [2-({6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-yl}sulfanyl)acetamide] is the most potent and selective adenosine A2B receptor (A2B AR) agonist known to date. Therefore, it has been widely used for in vitro and in vivo experiments. In the present study, we investigated the binding and functional properties of BAY60-6583 in various native and recombinant cell lines with different A2B AR expression levels. In cAMP accumulation and calcium mobilization assays, BAY60-6583 was found to be significantly less efficacious than adenosine or the adenosine derivative NECA. When it was tested in human embryonic kidney (HEK)293 cells, its efficacy correlated with the A2B expression level of the cells. In Jurkat T cells, BAY60-6583 antagonized the agonistic effect of NECA and adenosine as determined in cAMP accumulation assays. On the basis of these results, we conclude that BAY60-6583 acts as a partial agonist at adenosine A2B receptors. At high levels of the physiologic agonist adenosine, BAY60-6583 may act as an antagonist and block the effects of adenosine at A2B receptors. This has to be considered when applying the A2B-selective "agonist" BAY60-6583 in pharmacological studies, and previous research results may have to be reinterpreted.