Calretinin is thought to play roles in calcium buttering. Its site of expression has been extensively studied in the central nervous system. We previously reported (Hong et at.,2002, Neurosci. Res.,44: 325-335) calretinin expression in the superficial layers of the cat superior colliculus (SC). In the present study, we studied the distribution of calretinin in the intermediate and deep layers by immunocytochemistry. We found striking differences in calretinin immunoreactivity among the superficial, intermediate, and deep layers. In contrast to the superficial layers, the intermediate and deep layers contained many calretinin-immunoreactive (IR) neurons. They formed two laminar tiers. The first tier, which was very distinctive, was found within the upper intermediate gray layers and formed clusters of labeled neurons in many sections. The second tier of calretinin-IR neurons was found in the deep gray layer. However, the second tier was not distinctive compared to the first tier and the labeled neurons did not form any clusters. Calretinin-IR neurons in the intermediate and deep layers varied dramatically in morphology and included vortical fusiform, pyriform, and stellate neurons. Neurons with varicose dendrites were also labeled. Most of the labeled neurons were small to medium in size. Enucleation appeared to have no effect on the distribution of calretinin immunoreactivity in the contralateral intermediate and deep layers of the SC. The results indicate that calretinin is present in various neurons, at different locations. These results should be applicable for better understanding of the functional organization of the SC.