Seven day chick embryo cerebral cortical neurons cultured at low density (10,000 cells/16 mm well) in serum-free, defined medium in polylysine-coated wells fail to extend neurites and assume a flattened phase-dark morphology. Addition of serum-free, defined medium conditioned over chick embryo heart cells promotes neurite outgrowth and rounding of the cell body (which becomes phase bright). A sensitive bioassay, based on counting the number of phase-bright neurons with processes at least equal to one cell body diameter after 20 hours in culture, was used to titrate neurite-promoting activity in heart conditioned medium. This activity is completely destroyed by exposure to trypsin. As little as 2 micrograms/ml total conditioned medium protein elicits a half-maximal response in this bioassay.