ATP is an excitatory neurotransmitter that is stored and cosecreted with catecholamines from cells of the adrenal medulla. While the transport of catecholamines into chromaffin granule ghosts has been extensively characterized, there is little information on the mechanism of ATP transport into these structures. Here we show that ATP transport is driven by the electrical component of the electrochemical proton gradient created by the chromaffin granule membrane H+-ATPase, and that the accumulated nucleotide is released from the vesicles by inhibition of the H+-ATPase. GTP and UTP are also substrates for this transporter, distinguishing it from the mitochondrial ADP/ATP exchanger. Accumulation of ADP and ATP (rather than exchange with intravesicular ATP) is demonstrated by high pressure liquid chromatography measurements. The anion transport inhibitor 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (Ki = 27 microM) inhibits ATP transport, while atractyloside, the inhibitor of the mitochondrial ATP/ADP exchanger, is a very poor inhibitor. Finally, we have demonstrated a synergy between the accumulation of ATP and that of serotonin (i.e. more of each solute accumulates when the two are accumulated together), supporting the view that there is an interaction between serotonin and ATP that reduces their effective concentration within the ghosts.