Intravenously administered interleukin-6 (IL-6), a monokine produced by activated monocytes and folliculostellate cells of the pituitary gland, has been recently reported to elevate plasma ACTH level and to stimulate PRL, GH and LH release from cultured pituitary cells. To determine the site(s) of action of IL-6 in the control of pituitary hormone release, we injected human recombinant IL-6 into the third brain ventricle (3V) of freely moving, conscious male rats. Both 0.05 and 0.25 pmol doses of IL-6 were ineffective to change plasma ACTH in comparison to the values in controls. The maximal IL-6 dose tested of 1.25 pmol increased plasma ACTH within 15 min and the response lasted over 180 min. Plasma TSH levels were significantly lowered by a dose of 0.25 pmol IL-6, but neither the lower dose of 0.05 pmol nor the higher dose of 1.25 pmol altered plasma TSH levels throughout the 180 min of the experiment. Plasma PRL and GH levels were not changed by any IL-6 dose tested. In ovariectomized rats plasma LH and FSH levels were also unaltered by IL-6. The effects of IL-6 on plasma ACTH and TSH were only partially paralleled by increased rectal temperature which suggests that hypothalamic temperature regulating centers were independent of these actions. To evaluate a possible direct effect on the pituitary, IL-6 was incubated in vitro with hemipituitaries under an atmosphere of 95% O<sub>2</sub>/5% CO<sub>2</sub>. After 1 h of incubation IL-6 failed to cause any change in the secretion of pituitary hormones throughout a concentration range of 10<sup>–15</sup>–10<sup>–9</sup> M. Increased ACTH and GH secretion into the incubation medium was found only with 10<sup>–13</sup> M IL-6 after a 2-hour incubation, whereas there was no effect on PRL, TSH, LH and FSH release. The results support a possible role for IL-6 at both hypothalamic and/or pituitary levels to stimulate ACTH and GH and to decrease TSH release.