Using optical imaging, the effects of excitatory amino acids and their antagonists on the spatial pattern of activity evoked by the stimulation of parallel fibers in the cerebellar cortex of the anesthetized rat were examined. A vermal folium was stained with the stryrl voltage-sensitive dye RH-795 and imaged with a cooled charge-coupled service system during the activation of the parallel fibers with surface stimulation. Stimulation of the cerebellar cortex produced discrete "beams" of optical activity consistent with extracellular field recordings. The signal-to-noise ratio was excellent (> 10:1), reducing the number of stimuli, exposure time, and acquisition time needed to produce images. Extracellular field potential recordings were used to assay neuronal activity as well as the effects of the various excitatory amino acid agonists. Application of several glutamate receptor antagonists reversibly blocked the optical signals as well as the synaptic components of the extracellular field potentials. Neither N-methyl-d-aspartate nor its antagonist, (±)-2-amino-7-phosphonoheptanoic acid, had any affect on the optical signals or field potentials. These results indicate that the optical signal is not due to the evoked parallel fiber activity, but is generated mainly by postsynaptic targets and the parallel fiber synaptic action is primarily mediated by non-N-methyl-d-aspartate receptors. Other excitatory amino acid agonists had a differential effect on the optical response. Glutamate and kainate increased the "on beam" optical signal evoked by parallel fiber stimulation, in amplitude and width. In contrast, quisqualate always decreased the amplitude and width of the optical beam. Also, quisqualate produced an increase in fluorescence lateral to the optical beam, possibily due to an increase in "off beam" inhibitory activity. The changes in the extracellular field potentials were in agreement with the effects on the optical signals. Two possible mechanisms are proposed to account for the inhibitory effect of quisqualate. One is that quisqualate desensitizes Purkinje cell receptors, the other is that inhibitory interneurons in the cerebellar cortex are more preferentially excited with quisqualate application which in turn inhibits Purkinje cells both "on beam" and "off beam". In conclusion, voltage-sensitive dye optical signals evoked by stimulation of the cerebellar surface were imaged at high signal-to-noise levels using a cooled charge-coupled device system. Use of excitatory amino acid agonists and antagonists demonstrated that the optical signal was dependent on postsynaptic activity and confirmed that the parallel fiber postsynaptic action is primarily mediated by non-N-methyl-d-aspartate receptors. The reported differential action of glutamate, quisqualate and kainate indicates how different excitatory amino acid receptor classes are involved in the control of spatial activity in the cerebellar cortex.