The pretectum is an important center for visual reflexes. However, the location, boundaries, and connections of individual nuclei of the pretectum are incompletely understood. In cat, the traditionally defined nuclear boundaries have been placed in doubt by recent evidence showing that the retinal input to the pretectum forms four continuous projection zones that do not match the cytoarchitectural boundaries of individual pretectal nuclei defined by previous studies. We now show that antibodies to the calcium-binding protein calbindin-D 28K (CaBP) label clusters of neurons within the pretectum that match the zones of retinal termination. Four obvious cell clusters within the pretectum were labeled by CaBP antisera. Computer three-dimensional reconstruction of these cell clusters revealed that they form four distinct but continuous zones that run the rostrocaudal length of the pretectum in a medial-to-lateral direction. By combining anterograde HRP labeling of retinal terminals with CaBP immunocytochemistry, these CaBP-labeled cell clusters were found virtually to overlap the retinal projection zones. The CaBP-labeled neurons included both multipolar and fusiform morphologies, and most were medium- to large-sized cells. HRP retrograde transport studies showed that many CaBP-labeled neurons in the clusters projected to the LGN, while none projected to the inferior olive (IO). GABA- immunoreactive neurons were also found within the CaBP cell clusters, but these neurons were smaller than most CaBP-labeled neurons, and none were retrogradely labeled following HRP injections into the LGN or IO. Two-color antibody double-labeling experiments did not reveal any GABA neurons within the clusters that colocalized CaBP. In summary, calbindin is a precise marker of neuron clusters that overlap the retinal projection zones in the cat pretectum. Many of these CaBP neurons project to the LGN, and none contain GABA.