Glycosaminoglycans (GAGs), localized on the surfaces of cells and in the basement membrane, modulate the growth and differentiation of many cell types. Recent studies have shown that heparin, a GAG found in mast cells, potentiates the ability of acidic fibroblast growth factor (aFGF) to induce neurite outgrowth in pheochromocytoma (PC12) cells. We examined the effect of a variety of GAGs on aFGF, basic fibroblast growth factor (bFGF), and nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. The effects observed were dependent upon the specific GAG, the concentration of the GAG, and the growth factor. Heparin potentiated aFGF-induced neurite outgrowth in a concentration-dependent fashion; potentiation increased with increasing heparin concentrations of 0.01-100 μg/ml. At concentrations greater than 100 μg/ml, heparin potentiation decreased. The maximally active concentration of heparin (100 μg/ml) increased the potency of aFGF 102-fold. Increasing concentrations of heparan sulfate, dermatan sulfate, and chondroitin sulfate correlated with increasing aFGF potentiation. The maximally active concentrations of heparan sulfate (100 μg/ml), dermata sulfate (10 mg/ml), and chondroitin sulfate (1 mg/ml) increased the activity of aFGF 11-, 110-, and 11-fold, respectively. Hyaluronic acid did not affect the neurite outgrowth-promoting activity of aFGF. Heparin also altered the activity of bFGF; increasing concentrations of heparin (0.01-1 μg/ml) correlated with increased potentiation. At concentrations greater than 1 μg/ml, heparin concentration was inversely correlated with potentiation. Chondroitin sulfate only increased the percentage of neurite-bearing cells at concentrations greater than 10 μg/ml. Maximally active concentrations of heparin (1 μg/ml) and chondroitin sulfate (1 mg/ml) increased the potency of bFGF 5-fold. The highest concentration of heparan sulfate studied (1 mg/ml) inhibited the activity of bFGF. Dermatan sulfate and hyaluronic acid (0.01–1000 μg/ml) had no effect on bFGF activity. Heparan sulfate and chondroitin sulfate showed concentration-dependent potentiation of NGF; maximally active concentrations of heparan sulfate (100 μg/ml) and chondroitin sulfate (1 mg/ml) increased the potency of NGF 3-fold, whereas heparin, dermatan sulfate and hyaluronic acid had no effect. None of the GAGs had any effect on PC12 neurite outgrowth when added alone. The specificity of the activity of the GAGs was verified by selective enzyme degradation. Chondroitin ABC lyase eliminated the potentiation observed with dermatan sulfate and chondroitin sulfate, but had no effect on heparin or heparan sulfate activity. Conversely, heparinase reduced the activity of heparin and heparan sulfate, but had no effect on the potentiation observed with dermatan or chondroitin sulfate. These data indicate that not only heparin, but also other sulfated GAGs found in the extracellular matrix, can alter the activity of neuronal growth factors in vitro and thus may also influence growth factor-mediated functions in vivo.