In vitro autoradiography was used to characterize and quantitatively map the sodium-independent binding of [3H]glutamate in rat brain. Measured in the presence of chloride, glutamate binding to frozen brain sections was specific, saturable, and reversible, with a Kd in the low micromolar range. At least two distinct binding sites were detected which had different affinities for quisqualic acid (0.7 PM and 1.2 mM). Autoradiograms revealed very high levels of binding in rostra1 forebrain areas, especially olfactory structures and frontal cortex.