The effects of chemical thyroidectomy produced by propylthiouracil treatment or hyperthyroidism produced by daily injections of thyroxine on the content of serotonin, its metabolite 5-hydroxyindoleacetic acid and of substance P in discrete brain nuclei of adult rats have been studied. Brain nuclei were removed by punches of frozen brain slices. The serotonin and 5-hydroxyindoleacetic acid contents were measured by high performance liquid chromatography with electrochemical detection while substance P was assayed by radioimmunoassay. The serotonin level was significantly increased in 11 nuclei of the hypothyroid and in 12 nuclei of the hyperthyroid rats. The 5-hydroxyindoleacetic acid levels were higher in 16 nuclei of the hypothyroid and 7 nuclei of the hyperthyroid animals. The state of the thyroid gland in adult rats also affected the substance P system but less consistently than the serotonin pathway. Thus, the substance P content in the brain of propylthiouracil-treated animals increased significantly in 4 nuclei while it decreased in the septum lateralis and the striae terminalis. Following the chronic thyroxine injections, the substance P level was increased in the nucleus caudatus putamen and the tractus diagonalis and was decreased in the area ventralis tegmenti. We previously reported that neonatal thyroidectomy caused a general increase of the substance P and serotonin contents in many brain nuclei of young rats. It appears that the effects of chemical thyroidectomy on mature and immature rat brain are different for the substance P system, suggesting that thyroxine plays an important role in the maturation of substance P-containing neurons.