Adenosine is an important neuromodulator in the brain. Traditionally, adenosine is thought to arise in the extracellular space by either an extracellular mechanism, where it is formed outside the cell by the breakdown of released ATP, or an intracellular mechanism, where adenosine made inside the cell is transported out. Recently, a proposed third mechanism of activity dependent adenosine release has also been proposed. Here, we used fast-scan cyclic voltammetry to compare the time course and mechanism of adenosine formation evoked by either low- or high-frequency stimulations in striatal rat brain slices. Low-frequency stimulations (5 pulses at 10 Hz) resulted in an average adenosine efflux of 0.22 ± 0.02 μM, while high-frequency stimulations (5 pulses, 60 Hz) evoked 0.36 ± 0.04 μM. Blocking intracellular formation by inhibiting adenosine transporters with S-(4-nitrobenzyl)-6-thioinosine (NBTI) or propentofylline did not decrease release for either frequency, indicating that the release was not due to the intracellular mechanism. Blocking extracellular formation with ARL-67156 reduced low-frequency release about 60%, but did not affect high-frequency release. Both low- and high-frequency stimulated release were almost completely blocked by removal of calcium, indicating activity dependence. Reducing dopamine efflux did not affect adenosine release but inhibiting ionotropic glutamate receptors did, indicating that adenosine release is dependent on downstream effects of glutamate. Therefore, adenosine release after short, high-frequency physiological stimulations is independent of transporter activity or ATP metabolism, and may be due to direct release of adenosine after glutamate receptor activation.