We have investigated the pattern of glutamate-like immunoreactivity in the superficial layers of the rat superior colliculus by means of postembedding immunocytochemical methods for light and electron microscopy. At the light microscopic level, labelling was faintly to moderately intense in most perikarya of the stratum zonale, stratum griseum superficiale and stratum opticum. Furthermore, strong glutamate-immunoreactive terminal-like elements were accumulated most densely in stratum zonale, stratum griseum superficiale and stratum opticum. At the electron microscopic level, a postembedding immunogold method revealed that the vast majority of those labelled elements corresponded to retinal and visual cortical terminals. These profiles were about twice as heavily labelled as their postsynaptic partners. To determine the contribution of retinal and cortical afferents to the pattern of glutamate-like immunoreactivity, rats were subjected to right retinal ablation, left cortical ablation or combined right retinal and left cortical ablations. After retinal ablation, strongly labelled perikarya were observed in the retinorecipient layers. Furthermore, a prominent loss of glutamate-immunoreactive terminal-like elements occurred in stratum zonale and stratum griseum superficiale. Ipsilateral superior colliculus to cortical ablation exhibited subtle changes characterized by a moderate increase in perikaryal immunostaining in stratum zonale, stratum griseum superficiale and stratum opticum and by an apparent discrete reduction of labelled dots in stratum griseum superficiale and stratum opticum. In cases with combined lesions, strongly immunoreactive cell bodies and dendrites were accompanied by a massive disappearance of labelled terminal-like elements in stratum zonale, stratum griseum superficiale and stratum opticum. The effect of retinal and visual cortical ablations on the pattern of glutamate-like immunoreactivity suggests that these afferents are the major sources for glutamate-immunoreactive terminals in the rat superior colliculus. In addition, these findings provide further evidence for glutamate as neurotransmitter in the visual pathways studied.