An in vitro receptor binding assay, using filtration to separate bound from free [125I]insulin, was developed and used to characterize insulin receptors on membranes isolated from specific areas of rat brain. The kinetic and equilibrium binding properties of central receptors were similar to those of hepatic receptors. The binding profiles in all tissues were complex and were consistent with binding in multiple steps or to multiple sites. Similar binding properties were found among receptors in olfactory tubercle/bulb, cerebral cortex, hippocampus, striatum, hypothalamus, and cerebellum. High affinity [125I]insulin binding sites (KD = 3-11 nM) were distributed evenly between membranes isolated from P1 and P2 fractions of these brain areas, with the exception of the olfactory tubercle in which binding to P2 membranes was four-fold greater (Bmax = 150 fmol/mg protein). One difference between insulin receptors in brain and peripheral target tissues, however, was observed. Following exposure to 0.17 microM insulin for 3 h at 37 degrees C, the number of specific [125I]insulin binding sites on adipocytes decreased by 40%, while the number of binding sites on minces of cerebral cortex/olfactory tubercle remained constant. The results suggest that although the binding characteristics of central and peripheral insulin receptors are similar, these receptors do not appear to be regulated in the same manner.