Abstract. Regulation of renal apical Na ϩ /H ϩ exchanger 3 (NHE3) activity by adenosine has been suggested to contribute to acute control of mammalian Na ϩ homeostasis. The mechanism by which adenosine controls NHE3 activity in a renal cell line was examined. The adenosine analog, N 6 -cyclopentyladenosine (CPA) exerts a bimodal effect on NHE3: CPA concentrations Ͼ10 Ϫ8 M inactivate NHE3, whereas concentrations Ͻ10 Ϫ8 M stimulate NHE3 activity. Acute CPA-induced control of NHE3 was blocked by antagonists of A 1 adenosine receptors and inhibition of phospholipase C, pretreatment with BAPTA-AM (chelator of cellular calcium), and exposure to pertussis toxin. Stimulatory and to some extent also inhibitory CPA concentrations attenuated 8-bromo-cAMP and dopaminemediated inhibition of NHE3. BAPTA eliminated the ability of a stimulatory dose of CPA to attenuate 8-bromo-cAMP-induced suppression of NHE3 activity. Upon inhibition of protein kinase C, CPA at an inhibitory dose provoked activation of NHE3, which is partially reverted by 8-bromo-cAMP and suppressed by pre-incubation with BAPTA-AM. Cytochalasin B, an actin-modifying agent, selectively prevented downregulation but did not affect upregulation of NHE3 activity by CPA. In conclusion, these observations demonstrate that (1) CPA modulates NHE3 activity by elevation of cellular Ca 2ϩ exerting a negative control on adenylate cyclase activity, (2) protein kinase C is the determining factor leading to CPAinduced downregulation of NHE3 activity, and (3) alterations of surface NHE3 abundance may contribute to A 1 adenosine receptor-dependent inhibition of NHE3 activity.