We have examined the effect of insulin and tunicamycin, which cause decreases in cell surface insulin receptor numbers in peripheral tissues, on insulin receptors in neuron-enriched brain cell cultures. Incubation with 0.016-0.83 microM insulin for 24 h failed to decrease the specific binding of 125I-insulin in neuron-enriched cultures prepared from whole brains of 1-day-old rats. In contrast, these concentrations of insulin produced a dose-dependent decrease in the binding of 125I-insulin in fibroblastic cultures. Tunicamycin, an antibiotic which inhibits glycosylation of proteins and greatly reduced insulin binding via a reduction in apparent receptor numbers in fibroblastic cultures, caused no decrease in the binding of 125I-insulin in neuron-enriched cultures. These results indicate that brain insulin receptors are not down regulated by insulin and that glycosylation of insulin receptors may not be an important step in the regulation of their surface expression in the brain.