Neuronal cells from 1-day-old rat brain in primary culture have been utilized in the present study to characterize insulin-binding sites and a possible action of insulin on these cells. Binding of 125I-insulin to neuronal cultures was 90% specific and time-dependent and reached equilibrium in 120 min. Specific binding was reversible with greater than 90% of binding dissociable within 120 min with a t1/2 of dissociation of 15 min. Various insulin analogues competed for 125I-insulin binding to neuronal cultures according to their known biological potencies. Scatchard analysis of competition data yielded a typical curvilinear plot providing a class of high affinity (Kd = 11 nM) and low affinity (Kd = 65 nM) binding sites. Light microscopic autoradiographic analysis of 125I-insulin bound to neuronal cultures revealed the presence of silver grains predominantly on the neurites with occasional occurrence on the cell soma. Insulin had no effect on neuronal 2-deoxyglucose uptake in contrast with our previous findings demonstrating a 2-fold stimulation of 2-dGlc uptake into astrocyte glial cells from rat brain (Clarke, D.W., Boyd, F.T., Jr., Kappy, M.S., and Raizada, M. K. (1984) J. Biol. Chem. 259, 11672-11675). Incubation of neuronal cultures with insulin caused a dose-dependent inhibition of [3H]norepinephrine uptake with significant inhibition occurring at 1.67 X 10(-11) M. These findings demonstrate that: 1) neuronal cells in primary culture possess specific insulin receptors which are predominantly located on neurites and 2) insulin modulates monoamine uptake in these cultures which suggests that insulin may modulate neural signaling via specific neuronal insulin receptors.