Cultured corneal epithelial cells release a factor(s) that stimulates trigeminal neurons to form neurites in vitro. To characterize this trophic effect, conditioned media (serum free, supplemented) from cultures for corneal epithelium, stromal fibroblasts, and endothelium were studied further. Only epithelial conditioned medium (PCM) prolonged neuronal survival and induced neurite outgrowth. This trophic influence peaked after 2 to 3 days and gradually declined thereafter during a week when the medium was not renewed. Using a bioassay to score the percentage of initially viable neurons that extended neurites, it was found that the trophic effect of PCM was proportional to the conditioned medium concentration and to the cell density of the epithelial culture used for the conditioning. Maximum activity in PCM was correlated with confluency of the epithelial culture. Experiments using antiserum to nerve growth factor (NGF) and purified antibody to cold-insoluble globulin (CIG) indicated that the tropic effect of PCM was not derived from NGF or CIG. The trophic activity of PCM was abolished totally by heat or trypsin treatment but was not affected by collagenase. Although a fraction of the trophic activity was associated with the substratum after adsorption of PCM, this and other evidence did not suggest that the primary action of PCM was to enhance neuronal adhesion.