The binding of [125I]secretin to rat brain membranes was investigated. Radiolabeled secretin bound with high affinity (KD = 0.2 nM) to a single class of noninteracting sites. Binding was specific, saturable, and reversible. Regional distribution studies indicated that the specific binding was greatest in the cerebellum, intermediate in the cortex, thalamus, striatum, hippocampus, and hypothalamus, and lowest in the midbrain and medulla/pons. Pharmacological studies indicated that only secretin, but not other peptides, inhibits binding of [125I]secretin with high affinity. Also, certain guanine nucleotides inhibited high affinity binding. These data indicate that rat brain membranes possess high affinity binding sites specific for secretin and that with the use of [125I] secretin the kinetics, stoichiometry, specificity, and distribution of secretin receptors can be directly investigated.