Perturbations of interaction of hyaluronan (HA) with its receptor CD44 cause multiple errors in axon routing at the mouse optic chiasm. To investigate this interaction further on the chiasm routing, we studied the axon routing after enzymatic removal of HA from slice preparations of the optic pathway. Hyaluronidase treatment produced an obvious reduction in midline crossing of the first generated axons in E13 chiasms, but had no influence on routing ofthe uncrossed axons in E15 and E16 slices. These findings support a direct role of HA, acting probably through CD44, on axon decussation during early phase of chiasm development, but argue against a direct function of HA on the turning of uncrossed axons in the mouse optic chiasm.