We previously showed the selective expression of the chondroitin sulfate proteoglycans versican V0 and V1 in barrier tissues that impede the migration of neural crest cells during embryonic trunk development (Landolt, R. M., Vaughan, L., Winterhalter, K. H., and Zimmermann, D. R. (1995) Development 212, 2303-2312). To test for an active involvement of these isoforms in the guidance process, we have now established protocols to isolate intact versican V0 and V1 in quantities sufficient for functional experiments. Using stripe choice assays, we demonstrate that pure preparations of either a mixture of versican V0/V1 or V1 alone strongly inhibit the migration of multipotent Sox10/p75NTR double-positive early neural crest stem cells on fibronectin by interfering with cell-substrate adhesion. We show that this inhibition is largely core glycoprotein-dependent, as the complete removal of the glycosaminoglycan chains has only a minor effect on the inhibitory capacity. Our findings support the notion that versican variants V0 and V1 act, possibly in concert with other inhibitory molecules such as aggrecan and ephrins, in directing the migratory streams of neural crest cells to their appropriate target tissues.