Excitatory amino acids, acting at both N-methyl-D-aspartate (NMDA) and non-NMDA receptors, release the inhibitory neuromodulator adenosine from superfused rat cortical slices. This study was initiated to investigate the possible purinergic sources and mechanisms of release for the adenosine release evoked by NMDA and non-NMDA receptor activation. Inhibition of the bidirectional nucleoside transporter with dipyridamole greatly enhanced adenosine release evoked by glutamate. NMDA, kainate, and (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Inhibition of ecto-5'-nucleotidase with alpha, beta-methylene ADP and GMP had no effect on either kainate- or AMPA-evoked adenosine release, but it decreased glutamate- and NMDA-evoked adenosine release by 23 and 68%, respectively. A similar inhibition of NMDA-evoked adenosine release was observed with alpha, beta-methylene ADP alone, indicating that the inhibitory effect was not due to the reported competitive inhibition of NMDA receptors by GMP. Finally, NMDA-evoked adenosine release, but not kainate- or AMPA-evoked release, was Ca2+ dependent. These results indicate that activation of non-NMDA receptors releases adenosine per se in a Ca(2+)-independent manner. In contrast, NMDA receptor activation releases primarily a nucleotide that is subsequently converted extracellularly to adenosine; in this case, release is Ca2+ dependent. Although neither NMDA- nor non-NMDA-evoked adenosine release occurs via the nucleoside transporter, this transporter does appear to be a major route for removal of adenosine from the extracellular space.