The L-[3H]glutamic acid binding capability of rat cerebellar membranes prepared with or without preincubation at 37 degrees C followed by washing was investigated. The two preparations (KD = 820 nM, Bmax = 54.5 pmol/mg protein; KD = 509 nM, Bmax = 13.0 pmol/mg protein) showed no difference in specificity of the binding of the ibotenic acid analogues, consistent with the removal of an endogenous inhibitor by the preincubation at 37 degrees C followed by washing. The order of potency of the ibotenic acid analogues as inhibitors of L-[3H]glutamic acid binding is different from the order of potency in vivo, suggesting that the binding sites found are different from the physiological glutamic acid receptor.