In brain, levels of adenosine increase up to 100-fold during cerebral ischemia. Based on in vitro studies, both astrocytes and neurons contribute to this adenosine release. Neurons release adenosine per se whereas astrocytes release adenine nucleotides that are metabolized to adenosine extracellularly. In contrast, inosine is released from both cell types via a nucleoside transporter. C6 glioma cells, which are derived from astrocytes, release inosine but not adenosine. The present study investigated the relative expression of purine metabolizing enzymes and transporters in neurons, astrocytes and C6 glioma cells by real-time PCR analysis. In agreement with the extracellular formation of adenosine and intracellular formation of inosine by astrocytes, the present study showed high expression of ecto 5'-nucleotidase and AMP deaminase type 3 in astrocytes. The lack of adenosine release from C6 glioma cells was consistent with the absence of expression of the AMP-preferring cytosolic 5'-nucleotidase in these cells. The predominance of nitrobenzylthioinosine (NBMPR) insensitive equilibrative nucleoside transport (ENT2) in all three cell types was consistent with the greater activity of this isoform in comparison to NBMPR-sensitive ENT1 in these rat cells. Thus, cell type differences in adenosine formation and release are primarily a function of differences in expression of purinergic enzymes and transporters.