AbstractAim: To assess if adenosine is a direct growth hormone secretagogue receptor (GHSR) agonist by investigating the mechanism behind adenosine induced calcium release in human embryonic kidney 293s (HEK) cells expressing GHSR.Methods: Calcium mobilization, cyclic adenosine monophosphate (cAMP) and IP3experiments were performed using HEK cells stably expressing GHSR and/or adenosine A2Breceptor (A2BR).Results: Adenosine has been widely reported as a GHSR agonist. In our hands, adenosine and forskolin stimulated calcium release from IP3controlled stores in HEK–GHSR cells but not in non‐transfected HEK cells. This release was not accompanied by increased IP3levels. The calcium release was both cholera toxin and U73122 sensitive, indicating the involvement of both Gαs/adenylyl cyclase and Gαq/11/phospholipase C pathways. Importantly, the GHSR inverse agonist [D‐Arg1D‐Phe5D‐Trp7,9Leu11]‐Substance P (SP‐analogue) blocked the adenosine stimulated calcium release, demonstrating that GHSR is involved. Assessment of the GHSR‐dependent calcium release using adenosine receptor agonists and antagonists resulted in a rank order of potencies resembling the profile of A2BR. A2BR over‐expression in HEK–GHSR cells enhanced potency and efficacy of the adenosine induced calcium release without increasing IP3production. Moreover, A2BR over‐expression in HEK cells potentiated NECA‐induced cAMP production. However, GHSR expression had no effect on intracellular cAMP production.Conclusion: In HEK–GHSR cells adenosine activates endogenously expressed A2BR resulting in calcium mobilization. We hypothesize that the responsible mechanism is cAMP‐dependent sensitization of IP3receptors for the high basal level of IP3caused by GHSR constitutive activity. Altogether, our results demonstrate that adenosine is not a direct GHSR agonist.