The ability of astrocyte extracellular matrix to stimulate axonal elongation was examined using an in vitro model system. Extracellular matrix (ECM) was derived from primary cultures of rat astrocytes or meningeal cells, or from a cell line of bovine aortic endothelial cells. The cells were grown in 35-mm tissue culture dishes for 24 h and then removed non-enzymatically, leaving ECM attached to the surface of the culture dishes. Subsequently, PC12 pheochromocytoma cells were seeded onto the ECM and de novo neurite outgrowth was measured. Within 24 h, the PC12 cells exhibited profuse neuritic outgrowth on ECM derived from astrocytes and endothelial cells, without addition of exogenous nerve growth factor. Over a period of 4 days, the neurites continued to elongate and branched extensively. Little or no neuritic outgrowth was observed from PC12 cells grown on uncoated culture dishes or on culture dishes treated with astrocyte-conditioned medium. Only a slight stimulation of neurite outgrowth was observed on meningeal cell-derived ECM. These results indicate that astrocyte ECM, as well as endothelial cell ECM, possesses one or more molecular factors that can stimulate and maintain de novo axonal elongation from PC12 cells. It is suggested that immature astrocytes secrete neurite-promoting factors as a component of the ECM which act to stimulate and possibly guide the growth of axons during in vivo development.