Adenosine receptors (ARs) have emerged as new drug targets. The majority of data on affinity/potency and selectivity of AR ligands described in the literature has been obtained for the human species. However, preclinical studies are mostly performed in mouse or rat, and standard AR agonists and antagonists are frequently used for studies in rodents without knowing their selectivity in the investigated species. In the present study, we selected a set of frequently used standard AR ligands, 8 agonists and 16 antagonists, and investigated them in radioligand binding studies at all four AR subtypes, A 1 , A 2A , A 2B , and A 3 , of three species, human, rat, and mouse. Recommended, selective agonists include CCPA (for A 1 AR of rat and mouse), CGS-21680 (for A 2A AR of rat), and Cl-IB-MECA (for A 3 AR of all three species). The functionally selective partial A 2B agonist BAY60-6583 was found to additionally bind to A 1 and A 3 AR and act as an antagonist at both receptor subtypes. The antagonists PSB-36 (A 1 ), preladenant (A 2A ), and PSB-603 (A 2B ) displayed high selectivity in all three investigated species. MRS-1523 acts as a selective A 3 AR antagonist in human and rat, but is only moderately selective in mouse. The comprehensive data presented herein provide a solid basis for selecting suitable AR ligands for biological studies.