Accumulating evidence indicates that extracellular nucleotides can influence solute and water transport in a variety of epithelia. Stimulation of apical P2 receptors in the renal proximal tubule inhibits bicarbonate reabsorption (Bailey, 2004), and nanomolar concentrations of ATP have been detected in proximal tubular fluid both in vitro (Wilson et al. 1999) and in vivo (Shirley et al. 2003). The source of ATP in tubular fluid remains speculative. The present study has explored the possibility that ATP is stored in proximal tubular cells as vesicles prior to its release. The antimalarial drug quinacrine has a high affinity for ATP, which, upon binding, fluoresces under exposure to UV light. These properties make it suitable for labelling intracellular ATP. In the present study, quinacrine was used to visualize potential intracellular stores of ATP in an immortalized cell line (WKPT-0293) derived from the S1 segment of the proximal tubule of Wistar-Kyoto rats (Woost et al. 1996). Cells were grown to confluence (2-3 days) and were rinsed in Dulbecco-phosphate buffered saline (D-PBS). Cultures were then incubated with 1 ml of either isotonic (280 mosmol/kg H₂O) or hypotonic (140 mosmol/kg H₂O) D-PBS for 15 min at 37^oC. During incubation, samples of the medium were taken at 1, 5 and 15 min, immediately centrifuged, and the supernatant snap frozen in liquid nitrogen. The luciferin-luciferase assay was used to determine ATP concentration. Immediately after the 15 min incubation period, the same cells were incubated with quinacrine (2mmol/l) in isotonic D-PBS, rinsed with isotonic D-PBS, then photographed. Intense granular fluorescence was observed in cells maintained under isotonic conditions. In cells that had been subjected to hypotonic exposure, granular fluorescence was profoundly reduced. Extracellular ATP concentrations (mean ± SEM) under isotonic conditions were 4.0 ± 1.5, 4.6 ± 1.3 and 4.4 ± 1.5 nmol/l (n = 7) at 1, 5 and 15 min, respectively. Corresponding values during hypotonic exposure were 15.6 ± 6.0, 19.1 ± 5.8 and 25.4 ± 7.2 nmol/l (n = 7). The rise in extracellular ATP and the reduction in granular fluorescence during hypotonic stimulation support the proposition that the fluorescence observed during incubation with quinacrine was attributable to ATP. The data suggest that proximal tubular epithelial cells may store ATP in the form of vesicles which, upon stimulation, can be released into the extracellular milieu.