Here, the extracellular interconversion of nucleotides and nucleosides was investigated in rat hippocampal slices and synaptosomes by an HPLC-UV technique. Adenosine 5'-triphosphate (ATP) was converted to adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), adenosine, inosine, and hypoxanthine in the slices, whereas ADP elicited parallel and concentration-dependent formation of ATP and AMP. The specific adenylate kinase inhibitor diadenosine pentaphosphate decreased the rate of decomposition of ADP and inhibited the formation of ATP. No substantial changes in the interconversion of ADP to ATP and AMP were found in the presence of dipyridamole, flufenamic acid, the P2 receptor antagonist pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid tetrasodium (PPADS), and the alkaline phosphatase substrate para-nitrophenylphosphate. Negligible levels of nucleotides were generated when uridine 5'-diphosphate (UDP), AMP or adenosine were used as substrates. Ecto-adenylate kinase activity was also observed in purified synaptosomes. In summary, we demonstrate the presence of an ecto-adenylate kinase activity in the hippocampus, which is a previously unrecognized pathway that influences the availability of purines in the central nervous system.