Our previous studies demonstrated that optic nerve regeneration in the goldfish is accompanied by significantly enhanced axonal transport of glycosaminoglycans (GAGs), with a particularly large increase in the transport of chondroitin 4-sulfate (C4S). We have further shown that inhibition of proteoglycan (PG) synthesis and transport with beta-xyloside impedes axonal outgrowth from regenerating retinal explants. These results suggest a role for PGs that contain C4S in the regeneration process. To begin to address the possible functions of C4S GAGs during regeneration of goldfish retinal axons, we evaluated the effect of exogenous C4S on axonal outgrowth from regenerating retina, explanted 7-14 days after optic nerve crush. Our results indicate that exogenous C4S added to the culture medium potentiates axonal outgrowth on both polylysine- and LN-containing substrata. At low C4S concentrations, this potentiation is more marked on substrata containing both polylysine and LN than on polylysine alone. These results obtained in vitro suggest that soluble C4S, whether arising after axonal transport and externalization or after release from nonneuronal cells, is a positive modulator of regenerative axonal outgrowth in vivo.