Distribution of substance P (SP)-positive fibers in the medial preoptic area (POM) of the rat and their origins were examined using indirect immunofluorescence. A very high density of SP-positive fibers was seen in the POM throughout its entire rostro-caudal extent. However, the distribution of these fibers was not even; the highest density was detected in the medial part of the POM, with less dense but still numerous fibers in the lateral part. On the other hand, in this area a small number of SP-positive cells could be found; a few cells were scattered in the rostral part and, in the caudal part, several cells could be seen in the ventral part of the POM. The origins of SP-positive fibers in the POM were experimentally examined. Since the destruction of the ventro-lateral part of the anterior hypothalamus (vlAH), where numerous SP-positive cells were seen, resulted in a marked decrease of SP-positive fibers in the POM on the operated side, the majority of these fibers may originate from SP-positive cells in the vlAH. The fine structure of SP-positive terminals in the POM were investigated by electron-microscopic immunohistochemical techniques. Immunoreactive terminals contained a few large granular vesicles together with numerous small vesicles, and they made synaptic contacts mainly with dendrites which were devoid of immunoreactive materials. Two different synaptic contacts could be distinguished: one asymmetrical (Gray's type I) and the other symmetrical (Gray's type II), with the latter being predominant.