The development of the cerebral cortex involves the specification of intrinsic circuitry and extrinsic connections, the pattern of inputs and outputs. To investigate the development of a major afferent input to the cortex, we studied the formation of thalamocortical connections in an organotypic culture system. Slices from the lateral thalamus of young rats were cocultured with slices from the visual cortex. Thalamocortical projections in vitro were examined anatomically with fluorescent dyes and physiologically with electrophysiological and optical recording techniques. Axons emerged from thalamic explants radially in all directions. The outgrowth of thalamic fibers and the course of the axonal trajectories were not influenced by the presence of the cocultured cortex. Only those thalamic axons that happened to grow toward the cortical slices invaded their target tissue. Thalamocortical projection cell in vitro had the characteristic morphology of thalamic relay neurons. Cells with the morphology of interneurons were present in thalamic explants, but these neurons did not project to the cocultured cortex. Thalamocortical axons in vitro terminated in their appropriate cortical target layer, formed axonal arbors, and made functional synaptic contacts. Such specific connections between thalamic neurons and their cortical target cells were established regardless of whether thalamocortical axons invaded the cortex from the white matter side or from the pial surface. These results suggest that thalamic projection neurons have an innate mechanism that allows them to recognize their cortical target cells. Thus, intrinsic factors play a significant role in the laminar specification of cortical connections during development.