The effect of N-methyl-d-aspartate (NMDA) on regulation of extracellular adenosine was investigated in rat forebrain neurons in culture. NMDA evoked accumulation of extracellular adenosine with an EC50 value of 4.8 ± 1.2 µm. The effect of NMDA was blocked by (+)-5-methyl-10,11-dihydro-5H-dibenzo [a, d] cyclohepten-5,10-imine hydrogen maleate indicating that NMDA receptor activation was involved. The NMDA effect was also blocked by chelation of extracellular Ca2+ indicating that influx of calcium was required. The nitric oxide–cyclic GMP signalling pathway was not involved, as nitric oxide synthase inhibitors were unable to block, and cGMP analogs were unable to mimic, the effect of NMDA. The source for extracellular adenosine was likely to be intracellular adenosine as the ecto-5′-nucleotidase inhibitor αβ-methylene-ADP was unable to block the effect of NMDA. One possible cause of intracellular adenosine accumulation might be NMDA receptor-mediated inhibition of mitochondrial function and ATP hydrolysis. We found that NMDA caused a concentration dependent depletion of intracellular ATP with an EC50 value of 21 ± 8 µm. NMDA also caused a significant decrease in adenosine kinase activity, assayed by two different methods. Consistent with the hypothesis that inhibition of adenosine kinase is sufficient to cause an increase in extracellular adenosine, inhibition of adenosine kinase by 5′-iodotubercidin resulted in elevation of extracellular adenosine. However, in the presence of a concentration of 5′-iodotubercidin that inhibited over 90% of adenosine kinase activity, exposure to NMDA still caused adenosine accumulation. These studies suggest that several possible mechanisms are likely to be involved in NMDA-evoked extracellular adenosine accumulation.