How developing axons in the corpus callosum (CC) achieve their homotopic projection to the contralateral cortex remains unclear. We found that axonal position within the CC plays a critical role in this projection. Labeling of nearby callosal axons in mice showed that callosal axons were segregated in an orderly fashion, with those from more medial cerebral cortex located more dorsally and subsequently projecting to more medial contralateral cortical regions. The normal axonal order within the CC was grossly disturbed when semaphorin3A/neuropilin-1 signaling was disrupted. However, the order in which axons were positioned within the CC still determined their contralateral projection, causing a severe disruption of the homotopic contralateral projection that persisted at postnatal day 30, when the normal developmental refinement of contralateral projections is completed in wild-type (WT) mice. Thus, the orderly positioning of axons within the CC is a primary determinant of how homotopic interhemispheric projections form in the contralateral cortex.axon development | axon fiber order | cortical axon guidance | cortical development T he largest commissural tract in the human brain is the corpus callosum (CC), with more than 200 million axons connecting the two cerebral hemispheres. Callosal axons originate primarily from neurons of layer II/III and layer V of the neocortex (1) and project homotopically to the contralateral cortex. For example, callosal axons of the primary motor cortex (M1) and primary somatosensory cortex (S1) project to topographically equivalent locations in the contralateral M1 and S1, respectively. This pattern of homotopic projection is essential for coordinated motor and somatosensory functions as well as for higher associative and cognitive processes (2-4). Abnormal CC development has been noted in psychiatric and developmental disorders (5, 6), and deviant asymmetry of cortical areas found in patients with developmental dyslexia also may be attributed to callosal abnormalities (7,8). However, the mechanism by which normal homotopic projection pattern is achieved during development remains largely unknown.The majority of axonal projections in the nervous system are organized topographically. To facilitate the formation of orderly projections over long distances, axons originating from adjacent areas may preserve their topographic order within the nerve tract along their entire path toward the target region (9, 10). This preservation of topographic order has been shown in the thalamocortical tract (11)(12)(13)(14) and in the optic and olfactory nerves (15-17). By performing a series of random microinjections of biotinylated dextran amine in the dorsal thalamus and reconstructing the labeled fibers, Powell et al. (14) showed that labeled axons within the thalamocortical tract preserve a topography similar to that in the ventral telencephalon before they reach the cortex. In the developing olfactory system, axons within the olfactory nerve from three different regions of the olfactory epitheli...