This study of the tree shrew, Tupaia belangeri, provides evidence for an intracollicular pathway that arises in the superficial gray layer and terminates in the optic layer. As a first step, Nissl, myelin, and cytochrome oxidase stains were used to identify the layers of the superior colliculus in the tree shrew. Second, anterograde and retrograde axonal transport methods were used to determine relationships between laminar borders and patterns of connections. Intraocular injections of wheat germ agglutinin conjugated to horseradish peroxidase showed that the border between the superficial gray and optic layers in the tree shrew is marked by a sharp decrease in the density of retinotectal projections. The optic layer also could be distinguished from the subjacent intermediate gray layer by differences in connections. Of the two layers, only the intermediate gray layer received projections following injections of wheat germ agglutinin conjugated to horseradish peroxidase within substantia nigra pars reticulata. Similarly, following injections of horseradish peroxidase or biocytin in the paramedian pons, the intermediate gray but not the optic layer contained labeled cells of origin for the main premotor pathway from the tectum, the predorsal bundle. Next, cells in the superficial gray layer were intracellularly injected with biocytin in living brain slices. Axons were traced from narrow and wide field vertical cells in the deep part of the superficial gray layer to the gray matter surrounding the fiber fascicles of the optic layer. Small extracellular injections of biocytin in brain slices showed that the optic layer gray matter contains a population of stellate cells that are in position to receive the input from the superficial layer. Finally, small extracellular injections of biocytin in the intermediate gray layer filled cells that sent prominent apical dendrites into the optic layer, where they may be directly contacted by the superficial gray layer cells. Taken together, the results support the hypothesis that the optic layer is functionally distinct from its adjacent layers, and may provide a link in the transfer of information from the superficial, retinal recipient, to the intermediate, premotor, layer of the superior colliculus.