Neuroblastoma growth inhibitory factor (NGIF) exists in the conditioned medium of normal rat glioblasts. When neuroblastoma cells (Neuro2a, NS-20Y, and N1E-115) were cultured in the presence of the factor, the cell growth rates and DNA synthesis were markedly inhibited and the morphological differentiation including neural process formation was induced. However, the factor neither altered the growth rate nor the morphology of non-neuronal cells such as glial cell lines (C6 and 354A) or fibroblast (3T3). The molecular weight of the factor was estimated to be 75,000 Mr by gel filtration with Bio-Gel P-200, and the isoelectric point was 5.8. The factor was devoid of esteropeptidase activity, and susceptible to protease and thermal treatment. The growth inhibitory action of the factor was unrelated to the intracellular contents of cyclic AMP and GMP. The ability of NGIF to suppress preferentially the neural growth suggests its regulatory role in normal brain development.