Using an antibody against calbindin-28kD, we have studied the spatial pattern of expression of this protein in the superior colliculi (SC) of four strains of mature laboratory rats. In all four strains, calbindin-expressing cells (CECs) formed horizontally oriented tiers in the retinorecipient and intermediate gray layers but were diffusely distributed throughout the deep layers. Ontogenetically, calbindin-28kD was expressed for the first time in the retinorecipient layers at postconceptional day 20 (PCD 20), by cells located in the rostrolateral region where the first born retinal ganglion cells (RGCs) are represented. Although on the day of birth (PCD 22/23), the CECs were distributed more widely, they were still absent in the most medial part of the SC, that is, the region where the latest born RGCs are represented. The spatial distribution of CECs became adultlike only by PCD 29, that is, at the end of the period of the naturally occurring death of the RGCs. Monocular eye enucleations on PCD 23 prevented the expression of calbindin in the medial fifth of the retinorecipient layers of the contralateral SC, while the unilateral removal of the visual cortices had no discernable effect on the numbers and distribution of the CECs in either SC. Thus, the spatiotemporal pattern of ontogenetic expression of calbindin-28kD in the retinorecipient layers of SC reflects the spatiotemporal pattern of generation of the RGCs, and the retinal input appears to induce neuronal expression of calbindin-28kD in these layers.