The concentrations of Substance P and Neurokinin A were measured in plasma, and the hypothalamo-pituitary complex of 4-day-cycling female, ovariectomized and male rats. Estrous cycle-related fluctuations were recorded for these two neurokinins. The patterns of plasma concentrations of Substance P and Neurokinin A, however, were not similar throughout the rat estrous cycle. The plasma concentration of Substance P increased on proestrus at 19.00 hr, while Neurokinin A decreased. The plasma concentration of Substance P was positively correlated with plasma 17 beta-estradiol levels. In the ovariectomized rat, the absence of ovarian steroids led to low levels of plasma Neurokinin A, but the plasma concentration of Substance P did not show any change as compared to the estrous cycle. In the male rat, a similar observation was made in the presence of a testosterone environment. The largest variations in tissue concentration of both peptides were observed in the anterior pituitary. Substance P and Neurokinin A contents were higher throughout the proestrous day than the 3 other days. However, the level fell at 18.00 hr on proestrus, and there were similar falls in the hypothalamic contents of Substance P and Neurokinin A at 19.00 hr. In the ovariectomized rat, with no gonadal steroids, the hypothalamic and/or anterior pituitary levels of Substance P were in the same range as during the estrous cycle. However, the hypothalamic levels of Neurokinin A were lower and Neurokinin A was undetectable in the anterior pituitary. Substance P and Neurokinin A concentrations in the posterior pituitary were stable throughout the estrous cycle, with the exception of rises for both peptides on estrous day. Substance P levels were much lower in ovariectomized and male rats. These results describe large fluctuations in hypothalamic and pituitary Substance P and Neurokinin A contents through the estrous cycle in the female rat. They also strongly suggest the involvement of gonadal steroids in the differential regulation of Substance P and Neurokinin A in the female and male rat.