The hypothalamic neurons containing substance P (SP) were examined immunohistochemically in rats with a normal estrous cycle or persistent estrus (PE), in pregnancy and in lactating state. In PE animals, the neurons increased in number 1 week after ovariectomy, but then decreased and disappeared. During pregnancy, there were many SP neurons at an early stage but later they gradually decreased in number and disappeared. Consequently, during lactation no SP neurons were evident. The external layer of the median eminence (ME) contained many immunolabeled nerve terminals. In the ME prepared by a freeze-drying technique and immunostained with gold-labeled IgG by the postembedding procedure, the immunolabeled terminals were seen to contain large cored vesicles (50–100 nm) labeled with gold and unlabeled small clear vesicles (30–50 nm). In normal cycling rats, the terminals contained large cored vesicles in diestrus, small clear vesicles in estrus, and both in proestrus. In PE rats, the terminals contained only small clear vesicles, but the cored vesicles appeared after ovariectomy. In the early stage of pregnancy, the terminals contained only small clear vesicles, but the large cored vesicles appeared in late pregnancy and during lactation. These findings present further evidence that the hypothalamic SP neurons are involved in the hypothalamic-hypophysial-gonadal axis in r