The effect of adenosine regulation on sodium and chloride transport was examined in cultured A6 renal epithelial cells. Adenosine and its analogue N6-cyclopentyladenosine (CPA) had different effects on short-circuit current (Isc) depending on the side of addition. Basolateral CPA addition induced an approximately threefold increase of the Isc that reached a maximum effect 20 min after addition and was completely inhibited by preincubation with either an A2 selective antagonist, CSC, or the sodium channel blocker, amiloride. Apical CPA addition induced a biphasic Isc response characterized by a rapid fourfold transient increase over its baseline followed by a decline and a plateau phase that were amiloride insensitive. The A1 adenosine antagonist, CPX, completely prevented this response. This Isc response to apical CPA was also strongly reduced in Cl--free media and was significantly inhibited either by basolateral bumetanide or apical DPC preincubation. Only basolateral CPA addition was able to induce an increase in cAMP level. CPA, added to cells in suspension, caused a rapid rise in [Ca2+]i that was antagonized by CPX, not affected by CSC and prevented by thapsigargin preincubation. These data suggest that basolateral CPA regulates active sodium transport via A2 adenosine receptors stimulating adenylate cyclase while apical CPA regulates Cl- secretion via A1 receptor-mediated changes in [Ca2+]i.