The serotonergic innervation of the rat thalamus was studied with an indirect immunocytochemical technique using an antiserum raised against a serotonin-hemocyanin conjugate in animals pretreated with L-tryptophan and a monoamine oxidase (MAO) inhibitor. When pretreatment was not used there was a decrease in the number of immunoreactive fibers observed in the thalamic region. Innervation was greatest in nucleus ventralis corporis geniculati lateralis, and in the following midline nuclei: nucleus periventricularis, nucleus rhomboideus, and nucleus reuniens. Also well labeled were nucleus anterior ventralis, the intralaminar nuclei, and nucleus lateralis dorsalis. Moderate innervation was found in nucleus reticularis, nucleus anterior dorsalis, nucleus ventralis medialis, nucleus lateralis pars posterior, the posterior complex, and in nucleus dorsalis corporis geniculati lateralis. Very few serotonergic fibers were observed in nucleus paratenialis, nucleus gelatinosus, nucleus anterior medialis, nucleus medialis dorsalis, nucleus ventralis, nucleus ventralis pars dorsomedialis, or in nucleus corporis geniculati medialis. Serotonin immunoreactivity was also noted in a number of fiber bundles in the thalamic region. These include the fasciculus retroflexus, the fasciculus mamillothalamicus, the stria medullaris, and the stria terminalis. These results differ from those of previous descriptions of the serotonergic innervation of thalamic nuclei most notably in the midline nuclei and in the posterior complex. In this study the midline nuclei, nucleus rhomboideus, and nucleus reuniens were more densely innervated than had been described, and in the posterior complex a moderate, rather than sparse, innervation was observed. The more densely innervated nuclei of the anterior and lateral nuclear groups, nucleus lateralis dorsalis and nucleus anterior ventralis, also contained a greater number of labeled fibers than had been indicated.