Perturbation of the transmembrane glycoprotein, CD44, has been shown to cause multiple errors in axon routing in the mouse optic chiasm. In a recent report we have shown that the major CD44 ligand, hyaluronan (HA), is colocalized with CD44 at the midline of the chiasm, suggesting a possible contribution to the control of axon routing in the chiasm. We examined this issue by investigating the effects of exogenous HA on routing of axons in the chiasm in slice preparations of the optic pathway. In preparations of the E13 optic pathway, administration of exogenous HA produced a dose-dependent failure in midline crossing of the first generated optic axons. In E15 slices, when the adult pattern of axon divergence develops in the chiasm, anterograde filling of the optic axons showed an obvious reduction in the uncrossed pathway after HA treatment. This reduction was confirmed by retrograde filling of the ganglion cells in E15 slices, and later in E16 pathways where the uncrossed projection is better developed. Furthermore, we have demonstrated in explant cultures of the retina that HA, when presented in soluble or substrate-bound form, does not affect outgrowth and extension of retinal neurites. These findings together indicate the crucial functions of this matrix molecule in regulating midline crossing and axon divergence, probably through interactions with guidance molecules including CD44, at the midline of the chiasm.