Immunoreactive substance P (ir SP)-containing neurons were examined in rat hypothalamus. In untreated males, few if any ir perikarya were found in the arcuate nucleus, but after intraventricular colchicine administration, many appeared not only in the arcuate nucleus but also in the premammillary region, posterior hypothalamic, ventromedial, dorsomedial, subthalamic and paraventricular nuclei, and zona incerta. This was also the case in females treated with colchicine on the second day of diestrus. In untreated females, perikarya were seen only in the arcuate nucleus, varying in number and ultrastructure during the estrous cycle and being maximal in number in proestrus and estrus. The perikarya possessed well-developed Golgi bodies and lamellar bodies composed of many closely apposed cisternae of endoplasmic reticulum in proestrus, and showed stacks of two or three cisternae of endoplasmic reticulum as well as lysosomes in estrus. In diestrus, the perikarya had only a few granulated vesicles and fragmental cisternae of endoplasmic reticulum. In the perikarya of colchicine-treated males, many granulated vesicles and lysosomal bodies were noted. These findings suggest that, among the widely distributed SP neurons in the hypothalamus, some in the arcuate nucleus are involved in the hypothalamic-hypophysial-gonadal axis.