In adult rats, layer 5 cortical neurons send axons through the corpus callosum to contralateral cortex or through the internal capsule to subcortical targets, but individual neurons reportedly do not have both connections. Here we confirm this adult separation and address whether it develops by extension of axon collaterals to both sets of targets with later elimination of one or the other (a phenomenon common in developing cortex) or by initially selective axon outgrowth. Retrograde tracers Fast Blue and Diamidino Yellow were injected in the subcortical path at the pyramidal decussation and in the contralateral cortex, respectively, of adult and newborn rats. In 16 adults, no cortical neurons were double-labeled, indicating that none project to both sites. In 17 neonates, hundreds of thousands of layer 5 neurons were single-labeled in each brain, but only one was double-labeled. In cases in which the injections to one of the two targets was delayed, again, no double-labeled cells were found. These results indicate that the connectional distinction found in adults is not achieved by the elimination of long transient callosal or subcortical collaterals. To determine if shorter transient collaterals are extended by callosal neurons into the internal capsule, i.e., the subcortical pathway, we injected DiI into one cortical hemisphere of aldehyde-fixed Embryonic Day (E)19 and E21 brains. Two types of axons are seen in the white matter of the cortex opposite the injection: those which turn and extend through the callosum and those which branch, sending collaterals to both ipsilateral and contralateral cortex. Rarely were collaterals seen to extend into or toward the internal capsule. [3H]Thymidine birthdating studies show that layer 5 callosal and subcortically projecting neurons are generated at the same stage of corticogenesis. Thus, from early stages of axon extension, callosal and subcortically projecting cells are distinct classes of neurons and, responding differentially to cues present in cortex, initiate growth toward class-specific and nonoverlapping sets of targets. We conclude that the distinction between the two projection classes in inherent to them and is likely to be determined at an early stage of cortical development, prior to neuronal migration from the neuroepithelium.