We previously, described the production and secretion of insulin by fetal neurons in culture and demonstrated that neuronal synthesized insulin [I(n)] promoted neurofilament distribution and axonal growth. In this study we investigated the role of I(n) in promoting neural differentiation. Stem cells from 16 day gestational age rat brains were cultured in an insulin-free defined medium (IFDM) and treated with: 5, 20 or 100 ng/ml of exogenous insulin, 100 ng/ml insulin-like growth factor I (IGF-I) or an anti-insulin antibody. The neurons were studied at 1 and 3 days of incubation. The total number of cells showed no significant difference (P>0.05) in any of the media used, except the IFDM at day 3 of incubation treated with the anti-insulin antibody (P<0.05) and IGF-I to 20 ng/ml of insulin (P<0.05). No significant difference (P>0.05) was found in the number of differentiated neurons incubated in the IFMD, in which the neurons produce and secrete I(n), between days 1 and 3 of incubation, but neural differentiation decreased significantly (P<0.05) when treated with the anti-insulin antibody. Exogenous insulin significantly increased (P<0.05) the number of differentiated neurons compared to the IFDM. A significant reduction (P<0.05) of differentiated neurons was observed at day 3 of incubation with IGF-I compared to all the different media. Thus, I(n) has a role in promoting neural differentiation and growth, but exogenous insulin promoted neural differentiation and growth beyond I(n).