The neuropeptide, neurotensin, is localized to neurons within the hypothalamus which project to the median eminence. It is released from the terminals in the median eminence into the hypophyseal portal vessels and is carried to the gland. The content in the pituitary gland cells may be partly related to the delivery of the peptide via the portal vessels, but it also appears to be produced directly in pituitary cells. The peptide has actions on the release of glycoprotein hormones from the pituitary and in the present experiments, we attempted to determine whether these actions of the peptide were physiologically significant by microinjecting purified antiserum directed against neurotensin into the third cerebral ventricle or intravenously into conscious freely moving rats. Blood samples were withdrawn from an indwelling intra-right atrial catheter. In ovariectomized rats with high levels of plasma gonadotropins because of removal of ovarian steroid negative feedback, the intraventricular injection of the higher (3 µl) dose of neurotensin antiserum (NT-AS) induced a more than 2-fold increase in plasma LH within 2 h which was maintained until 3 h after the injection and returned to basal values in the 4th and 5th hour. The lower 1-µl dose was ineffective and there was no response to the control normal rabbit serum (NRS) injections into the third ventricle in this and the other experiments. When the animals were primed with estrogen and progesterone to mimic preovulatory conditions, the results were altered in that the lower 1-µl dose of NT-AS produced an increase in plasma LH apparent from 3 to 5 h following injection whereas the 3-µl dose was ineffective. The antiserum induced the opposite response of FSH to that previously described for LH in the ovariectomized animals, namely a significant decrease in plasma FSH following the higher but not the lower dose of NT-AS. In the ovariectomized estrogen, progesterone-primed rats (OEP rats), the antiserum had no effect on plasma FSH. The intraventricular injection of either the 1- or 3-µl dose of NT-AS induced a depression in plasma TSH in the ovariectomized rats apparent from 3 to 5 h after injection, whereas in the OEP rats, this suppression was significant from 1 to 5 h only with the higher 3-µl dose. Intravenous injection of the antiserum was ineffective to modify FSH or LH in either the ovariectomized or OEP animals; however, intravenous injection of 40 µl of NT-AS suppressed plasma TSH within 2 h in ovariectomized and intact male rats. There was no effect of NT-AS on the release of FSH, LH or TSH from overnight cultured dispersed pituitary cells of male rats. We conclude that neurotensin has a physiologically significant inhibitory action on the release of LH mediated at the hypothalamic level, presumably by suppressing the release of LHRH. On the other hand, at least in the ovariectomized rat, there is a physiologically significant hypothalamic action of the peptide to stimulate FSH release, illustrating the differential hypothalamic control of these two hormones. In contradistinction to these hypothalamic actions of the peptide, neurotensin appears to have a physiologically significant TSH-releasing action by direct stimulation of the thyrotropes.