Chondroitin sulfate (CS) proteoglycans have been implicated as molecules that are involved in axon guidance in the developing neural pathways. The spatiotemporal expression of CS was investigated in the developing retinofugal pathway in mouse embryos by using the CS-56 antibody. Immunoreactive CS was detected in inner regions of the retina as early as embryonic day 11 (E11). Its expression in subsequent stages of development followed a centrifugal, receding gradient that appeared to correlate with the sequence of axogenesis in the retina. In the chiasm, immunoreactive CS was expressed at E12, before the arrival of retinal axons. When the retinal axons navigated in the chiasm at E13-E14, immunoreactive CS remained at a low level in the optic fiber layer of the chiasm but was observed prominently in the caudal parts of the ventral diencephalon. This pattern followed closely the array of stage-specific-embryonic-antigen-1-positive neurons in the ventral diencephalon, with a V-shaped configuration that bordered the posterior boundary of the retinal axons, and a rostral raphe extension that ran across the decussating axons in the chiasm. Thus, the CS epitope is implicated in patterning the course of early retinal axons and in regulating axon divergence in the chiasm. At the lateral region of the chiasm, where the retinal axons cross the midline and approach the optic tract, a CS-immunopositive region coincided with the region in which active sorting of dorsal retinal axons from ventral retinal axons occurs. Moreover, at the threshold of the optic tract, the immunoreactive CS was restricted only to the deep part of the optic fiber layer, suggesting an inhibitory role of the CS epitope in repelling newly arrived axons to superficial regions of the optic tract during the development of chronotopic order at this part of the retinofugal pathway.