Glycosaminoglycans (GAGs), the carbohydrate moieties of proteoglycans, are thought to be positive and negative regulators of axonal growth. The physiological role of GAGs is controversial as some studies have shown that GAGs inhibit cell adhesion and neurite elongation (Exp Neurol 109:111, 1990) whereas other studies have reported a growth stimulatory effect of GAGs (Development 114:17, 1992). These and other studies have examined the effects of GAGs using different types of neurons and different substrate conditions thereby making a direct comparison of the experimental data difficult. To resolve the controversy concerning the ability of exogenous GAGs to modulate neurite growth, we examined the effects of a panel of structurally different GAGs on the growth of postnatal rat cerebellar granule neurons and embryonic rat dorsal root ganglia (DRG) neurons on substrates of either laminin or the L1 glycoprotein. Here we show that chondroitin 4-sulfate (CS4), chondroitin 6-sulfate (CS6), and keratan sulfate (KS) inhibit neurite growth from both cerebellar and DRG neurons on laminin-coated surfaces. On L1 surfaces, however, these GAGs are either extremely weak inhibitors of neurite extension or, in the case of CS4, a modest stimulator of neurite growth. Heparan sulfate (HS) and dermatan sulfate (DS) inhibited the growth of cerebellar neurons but not the growth of DRG neurons on L1-coated surfaces. On laminin surfaces, DS and HS had no effect on neurite growth from both cerebellar and DRG neurons. These results demonstrate a cellular and a substrate specificity to the effects of exogenous GAGs on neurite extension in vitro. They suggest that while CS and KS GAGs may not exert strong negative influences over axonal growth in regions of the developing CNS where the L1 glycoprotein is abundant, these GAGs are capable of inhibiting the growth of axons that extend within an environment rich in laminin.