A microbore column high-performance liquid chromatography (HPLC) system was used to measure neurotransmitters in tissue extracts and in vivo microdialysates obtained from the preoptic area (PO) and anterior hypothalamus (AH) of rats. The extracts contained norepinephrine, epinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC), dopamine, 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT), aspartate, glutamate, GABA, acetylcholine (ACh) and choline. The microdialysates obtained from the PO and AH of freely moving rats contained all of these substances except for norepinephrine, epinephrine, dopamine, and 5-HT. During collection of microdialysate from the PO and AH, core body temperature and locomotor activity were simultaneously measured by means of telemetry. The locomotor activity and body temperature increased during the night. This was accompanied by increased levels of 5-HIAA. The results suggest that serotonergic neuronal mechanisms in the PO and AH may be involved in hypothalamic regulation of spontaneous behaviors and body temperature.