Objective. We report the transcranial functional photoacoustic (fPA) neuroimaging of N-methyl-D-aspartate (NMDA) evoked neural activity in the rat hippocampus. Concurrent quantitative electroencephalography (qEEG) and microdialysis were used to record real-time circuit dynamics and excitatory neurotransmitter concentrations, respectively.Approach. We hypothesized that location-specific fPA voltage-sensitive dye (VSD) contrast would identify neural activity changes in the hippocampus which correlate with NMDA-evoked excitatory neurotransmission.Main results. Transcranial fPA VSD imaging at the contralateral side of the microdialysis probe provided NMDA-evoked VSD responses with positive correlation to extracellular glutamate concentration changes. qEEG validated a wide range of glutamatergic excitation, which culminated in focal seizure activity after a high NMDA dose. We conclude that transcranial fPA VSD imaging can distinguish focal glutamate loads in the rat hippocampus, based on the VSD redistribution mechanism which is sensitive to the electrophysiologic membrane potential.Significance. Our results suggest the future utility of this emerging technology in both laboratory and clinical sciences as an innovative functional neuroimaging modality.