Soluble chondroitin sulfate proteoglycans (CSPGs), prepared from 10-d- old rat brain, were added to the culture medium of PC12D cells containing NGF to examine the effects on NGF-induced neurite outgrowth from the cells. PC12D cells, a flat-shaped variant of PC12 pheochromocytoma cells, are characteristic of prompt neurite formation in response not only to NGF, but also to cAMP-enhancing reagents such as forskolin. Brain CSPGs inhibited the neurite elongation irreversibly in a dose-dependent manner; complete inhibition was observed at a concentration of 50 nmol uronic acid/ml. Closely similar dose-dependent inhibition was observed in the forskolin-induced neurite outgrowth from PC12D cells. NGF-induced neurite outgrowth from conventional PC12 cells was also inhibited completely by 50 nmol uronic acid/ml CSPGs. Some brain CSPGs seemed to be inhibitory, but the cartilage-unique CSPG did not show any inhibitory effect. Chondroitin sulfate, a polysaccharide moiety of CSPGs, did not show any inhibitory effect even at a concentration of 250 nmol uronic acid/ml, while core proteins prepared from brain CSPGs by digestion with chondroitinase ABC exhibited inhibitory activity similar to that of intact CSPGs. This indicates that the site of the inhibitory activity exists in the core protein moiety of brain CSPGs. From these observations, it is conceivable that brain CSPGs are involved in the regulation of neuronal differentiation.