Dopamine-glutamate interactions in the striatum are critical for normal basal ganglia-mediated control of movement. Although regulation of glutamatergic transmission by dopamine is increasingly well understood, regulation of dopaminergic transmission by glutamate remains uncertain, given the apparent absence of ionotropic glutamate receptors on dopaminergic axons in dorsal striatum. Indirect evidence suggests glutamatergic regulation of striatal dopamine release is mediated by a diffusible messenger, hydrogen peroxide (H₂O₂), generated downstream from glutamatergic AMPA receptors (AMPARs). The mechanism of H₂O₂-dependent inhibition of dopamine release involves activation of ATP-sensitive K⁺ (K_ATP) channels. However, the source of modulatory H₂O₂ is unknown. Here, we used whole-cell recording, fluorescence imaging of H₂O₂, and voltammetric detection of evoked dopamine release in guinea-pig striatal slices to examine contributions from medium spiny neurons (MSNs), the principal neurons of striatum, and dopamine axons to AMPAR-dependent H₂O₂ generation. Imaging studies of H₂O₂ generation in MSNs provide the first demonstration of AMPAR-dependent H₂O₂ generation in neurons in the complex brain-cell microenvironment of brain slices. Stimulation-induced increases in H₂O₂ in MSNs were prevented by GYKI-52466, an AMPAR antagonist, or catalase, an H₂O₂ metabolizing enzyme, but amplified by mercaptosuccinate (MCS), a glutathione peroxidase inhibitor. By contrast, dopamine release evoked by selective stimulation of dopamine axons was unaffected by GYKI-52466 or MCS, arguing against dopamine axons as a significant source of modulatory H₂O₂. Together, these findings suggest that glutamatergic regulation of dopamine release via AMPARs is mediated by retrograde signaling by diffusible H₂O₂ generated in striatal cells, including medium spiny neurons, rather than in dopamine axons.