LY334370 is a high affinity, selective agonist at the 5-HT(1F) receptor. On this basis, the tritiated compound was examined for its utility in autoradiography to localize the 5-HT(1F) receptor in rat and guinea pig brain regions. Specific 5-HT(1F) receptor binding in rat brain was found in layers 4-5 of all cortical regions examined, as well as olfactory bulb and tubercle, nucleus accumbens, caudate putamen, parafascicular nucleus of the thalamus, medial mammillary nucleus, the CA3 region of the hippocampus, subiculum, and several amygdaloid nuclei. In guinea pig brain, the [(3)H]LY334370 binding sites were found at highest density in claustrum, but also in a layer of the cortex, caudate putamen, nucleus accumbens, thalamus, and medial mammillary nucleus. Some species differences in the distribution of the 5-HT(1F) receptor were noted. Side by side comparison of rat brain autoradiography with [(3)H]LY334370 and [(3)H]sumatriptan showed labeling in the same brain regions. Preliminary binding studies in rhesus monkey and human brain sections showed [(3)H]LY334370 binding in cortical layers 4-5, subiculum (in the monkey), and the granule cell layer of the cerebellum. These findings suggest a discrete localization of the 5-HT(1F) receptor in the rat, guinea pig, monkey and human brain, and confirms the utility of [(3)H]LY334370 as a potential tool to explore further the localization and possible functions of the 5-HT(1F) receptor.