1. The effects of spiperone on 3H-5-HT binding to membranes prepared from rat diencephalon and lower brain stem were studied for comparison with telencephalic structures. 2. The neuroleptic spiperone produced shallow, complex inhibition curves of 3H-5-HT binding in the non-telencephalic areas, suggesting the presence of different types of high-affinity 3H-5-HT binding sites. However, the shape of the curves from the non-telencephalic areas differed from those of telencephalic regions. For example, there was a tendency for low concentrations of spiperone to cause an increase in binding in the diencephalon and lower brain stem rather than a decrease as seen in telencephalic areas. 3. In a search for other compounds which, like spiperone, could discriminate between the different types of 3H-5-HT binding sites in rat telencephalon, it was found that most drugs show little difference in their affinities for the two sites. However, one compound, AHR 1709B, was found to be about 10 times more potent in rat frontal cortex than corpus striatum, indicating that it can differentially recognize the two binding sites. 4. These results are consistent with the existence of multiple types of 3H-5-HT binding sites in the rat brain, and they define regional differences that had not previously been described. In addition, a new compound which can discriminate between different types of 3H-5-HT binding sites has been found, leading the way to the study of a series of new drugs which may selectively alter serotoninergic function.