The effect of afferent lesions on glutamate content was measured in the lamina of the superior colliculus (SC) in the rat. The analysis was performed 12 days after unilateral enucleation (left eye), or ablation of visual cortex (right), or both enucleation and ablation. The glutamate contained in the superficial grey layer (SGL) and deep layer was measured in the sectioned freeze-dried sample using an enzymatic cycling method of NAD-NADH. The upper layer of SGL contralateral to enucleation exhibited a significant reduction (23%) in glutamate content. Combining enucleation and ablation further decreased (35%) glutamate content. Additionally, the synaptic potential evoked in the SGL of SC slices after stimulation of optic layer was blocked by the application of kynurenic acid (3 mM) or DNQX (30 microM). These results indicate that the retino-tectal pathway in the rat can be glutamatergic in nature.