Extracellular ATP interacts with purinergic P2 receptors to regulate a range of physiological responses, including downregulation of transport activity in the nephron. ATP is released from cells by mechanical stimuli such as cell volume changes, and autocrine signaling by extracellular ATP could occur in renal medullary cells during diuresis. This was tested in MDCK cells, a model used frequently to study P1 and P2 receptor activity. ATP was released within 30 sec after transfer from 500 to 300 mosmol/kg medium. A 30 min incubation with ATP (0.01 to 0.10mM) produced dose-dependent inhibition of the renal betaine/GABA transporter (BGT1) with little effect on other osmolyte transporters. Inhibition was reproduced by specific agonists for P2X (methylene-ATP) and P2Y (UTP) receptors. Adenosine, the final product of ATP hydrolysis, also inhibited BGT1 but not taurine transport. Inhibition by ATP and adenosine was blocked by pertussis toxin and A73122, suggesting involvement of Gi proteins and phospholipase C in post-receptor signaling. Both ATP and adenosine (0.1 mM)produced rapid increases in intracellular Ca²⁺, due to mobilization of intracellular Ca²⁺ stores and Ca²⁺ influx. Blocking these Ca²⁺ increases with BAPTA-AM also blocked the action of ATP and adenosine on BGT1 transport. Finally, immunohistochemical studies indicated that inhibition of BGT1 transport may be due to endocytic accumulation of BGT1 proteins from the plasma membrane. We conclude that ATP and adenosine, through stimulation of phospholipase C and intracellular Ca²⁺, may be rapidly acting regulators of BGT1 transport especially in response to a fall in extracellular osmolarity.