To explore target sites for endogenous D-serine that are different from the glycine site of the N-methyl-D-aspartate (NMDA) type glutamate receptor, we have studied the binding of D-[3H]serine to the synaptosomal P2 fraction prepared from the rat brain and peripheral tissues in the presence of an excess concentration (100 microM) of the glycine site antagonist 5,7-dichlorokynurenate (DCK). Nonspecific binding was defined in the presence of 1 mM unlabeled D-serine. Association, dissociation, and saturation experiments indicated that D-[3H]serine bound rapidly and reversibly to a single population of recognition sites in the cerebellar P2 fraction in the presence of DCK, with a K(D) of 614 nM and a Bmax of 2.07 pmol/mg of protein. D-Serine, L-serine, and glycine produced a total inhibition of the specific DCK-insensitive D-[3H]serine binding to the cerebellum with similar Ki values. Strychnine and 7-chlorokynurenate failed to inhibit the binding at 10 microM. The profiles of displacement of the DCK-insensitive D-[3H]serine binding by various amino acids and glutamate and glycine receptor-related compounds differ from those of any other defined recognition sites. DCK-insensitive D-[3H]serine binding was at high levels in the cerebral cortex and cerebellum but very low in the kidney and liver. The present findings indicate that the DCK-insensitive D-[3H]serine binding site could be a novel candidate for a target for endogenous D-serine in mammalian brains.