Functional magnetic resonance imaging (fMRI) rests on the assumption that regional brain activity is closely coupled to regional cerebral blood flow (rCBF) in vivo. To test the degree of coupling, cortical brain activity was locally stimulated in rats by reversed microdialysis infusion of picrotoxinin, a γ-aminobutyric acid-A antagonist. Before and during the first 30 minutes of infusion, simultaneous fMRI (rCBF) and neurochemical (interstitial glutamate concentration) measures of brain activity were highly correlated (r = 0.83). After 30 minutes of picrotoxinin-induced stimulation, glutamate levels decreased but rCBF remained elevated, suggesting that additional factors modulate the relationship between neuronal neurotransmitters and hemodynamics at these later stages.Keywords: Microdialysis; Picrotoxin; Regional cerebral blood flow; Brain mapping