The distribution of serotonin binding sites was studied in the rat hippocampal region by using contact-film autoradiography after in vitro incubations of brain sections with 5-[3H]hydroxytryptamine, [3H]spiperone, and [3H]ketanserin, respectively. Biochemical studies of the 5-[3H]hydroxytryptamine binding to sections cut through the hippocampal region showed that at saturating concentrations of 5-[3H]hydroxytryptamine (2-2.5 nM) the specific binding was at least 50% of the total. The 5-[3H]hydroxytryptamine binding sites were found to be heterogeneously distributed within the hippocampal region with the highest densities present in the following parts: layers I and II and layers IV through VI of the entorhinal area, the radial layer of the subiculum and subfield CA1 of the Ammon's horn and the molecular layer of the area dentata. Moderate to low densities of binding was observed in layer III of the entorhinal area, the pre- and parasubiculum, the stratum pyramidale of the Ammon's horn, and the granular cell layer of the area dentata. Removal of the 5-hydroxytryptamine nerve terminals by systemic injections of the 5-hydroxytryptamine neurotoxin parachloroamphetamine resulted in no detectable reductions of 5-[3H]hydroxytryptamine binding in any brain region. Lesions of hippocampal cell bodies by intrahippocampal injections of ibotenic acid prevented the binding of 5-[3H]hydroxytryptamine within the area of the cell loss. Comparisons between the distribution of 5-hydroxytryptamine immunoreactive nerve terminals and the 5-[3H]hydroxytryptamine binding sites showed that in some areas of sparse 5-hydroxytryptamine innervation the 5-[3H]hydroxytryptamine binding was close to background (e.g. the pyramidal cell layer, the stratum lucidum) whereas in areas with little 5-[3H]hydroxytryptamine binding (e.g. layer III of the lateral entorhinal area, the presubiculum) a very dense 5-hydroxytryptamine innervation was found. The hippocampal 5-[3H]hydroxytryptamine binding was displaced neither by ketanserin (1 microM) nor by spiperone (1 microM), two drugs that bind to cortical 5-hydroxytryptamine2 receptors in the rat brain. Furthermore, the pattern of hippocampal [3H]spiperone binding differed considerably from that of 5-[3H]hydroxytryptamine. The [3H]ketanserin binding in the hippocampal region did not exceed background levels, except in the hilus of area dentata in the ventral hippocampus and entorhinal layer VI at the same level, where moderate binding was found.(ABSTRACT TRUNCATED AT 400 WORDS)