The localization and distribution of serotonin (5-HT) has in the rat brain been studied with the indirect immunofluorescence technique of Coons (1958) using a newly developed, specific antibody to 5-HT. This paper contains a detailed description of the distribution of 5-HT in the hypothalamus and hypophyis and a brief report on the localization of 5-HT containing cell bodies. Principally, 5-HT perikarya were present in neuron systems with a distribution similar to that revealed by the mapping of Dahlström & Fuxe (1964) with the formaldehyde-induced-fluorescence technique (FIF). However, in addition to the nine areas originally described, several other areas in the mesencephalon and rhombencephalon appeared to contain 5-HT cell bodies. In the hypothalamus no 5-HT positive neuronal perikarya could be observed. However, in the infundibulum and in the median eminence some 5-HT positive tanycytes and mast cells were observed. Serotonergic fibers and terminals are present throughout the hypothalamus. Particularly dense plexus of the fibers and terminals have been observed in the lateral hypothalamic area, nucleus mamillaris medialis, the nucleus perifornicalis, the nucleus suprachiasmaticus and the nucleus ventromedialis hypothalami. All remaining hypothalamic areas demonstrate various densities of 5-HT immunoreactive nerve fibers. In the pituitary 5-HT-like immunoreactivity have been found in the pars nervosa.