Immunocytochemistry and retrograde labeling were used to define the thalamic projections of calbindin- and parvalbumin-containing cells in superficial layers of the rat's superior colliculus (SC). Quantitative analysis revealed that 90.8 ± 2.2% (mean ± standard deviation) of the calbindin-immunoreactive neurons in the stratum griseum superficiale (SGS) projected to the dorsal lateral geniculate nucleus (LGNd) and that 91.3 ± 4.3% of calbindin-immunoreactive neurons in the stratum opticum (SO) projected to the lateral posterior nucleus (LP). In contrast, only 17.3 ± 2.5% of parvalbumin-immunoreactive neurons in the SGS were found to project to the LGNd and 16.5 ± 3.1% of the parvalbumin-immunoreactive SO cells were retrogradely labeled after LP injections. Few of the parvalbumin-immunoreactive neurons in either the SGS (7.2 ± 2.5%) or the SO (9.2 ± 2.5%) were GABA positive. The retrograde-labeling results suggest that parvalbumin-immunoreactive neurons in the rat's SO and SGS may either be primarily interneurons or have descending projections, while calbindin-containing cells are primarily thalamic projection neurons. These results are consistent with data from other rodents, but almost exactly the opposite of data that have been reported for the cat for these same populations of SC projection neurons. Such interspecies differences raise questions regarding the functional importance of expressing one calcium-binding protein versus another in a specific neuronal population.