We have developed a high specific activity radioiodinated ligand for the biochemical evaluation and autoradiographic localization of 5HT3 receptors in the brain. [125I]-(S)-iodozacopride was synthesized by radioiodination of deschloro-(S)-zacopride using chloramine-T, and the product was purified by HPLC. The equilibrium kinetics and pharmacology of the binding of this radioligand were studied in homogenates of rat cerebral cortex, while the distribution of binding was examined by quantitative autoradiography. [125I]-(S)-iodozacopride bound to a single, saturable, specific binding site (Kd = 192 +/- 9 pM, Bmax = 1.2 +/- 0.2 fmol/mg protein). The binding had the pharmacological properties of a 5HT3 receptor, being potently inhibited by a variety of 5HT3 agonists and antagonists including (S)-zacopride (Ki = 0.032 nM), Quipazine (Ki = 0.45 nM), LY278584 (Ki = 0.5 nM), (1-m-chlorophenyl)-biguanide (Ki = 0.6 nM) and ICS 205-930 (Ki = 1.0 nM). Autoradiographic studies were undertaken by incubating sections with 400 pM [125I]-(S)-iodozacopride and exposing them to film for 3-7 days to obtain suitable autoradiograms. Specific binding of [125I]-(S)-iodozacopride was found at various amounts in a variety of brain regions. The highest levels of binding were found in the brainstem, principally the nucleus of the solitary tract with somewhat lower levels in the area postrema, substantia gelatinosa of the trigeminal nucleus and dorsal motor nucleus of the vagus. In the rat forebrain, moderate levels of specific binding were found in the glomerular layer of the olfactory bulb, anterior olfactory nucleus and various subnuclei of the amygdala. Lower levels of binding were seen in the superficial laminae of the parietal cerebral cortex and diffusely distributed throughout the hippocampal formation. In conclusion, [125I]-(S)-iodozacopride binds to a receptor site with the pharmacological properties and distribution that is consistent with the 5HT3 receptor. [125I]-(S)-iodozacopride represents a significant improvement in autoradiographic studies of the 5HT3 receptor by reducing the required exposure time for producing autoradiograms from the 3-6 months required for [3H]-labeled ligands to 3-7 days.