Interferons (IFNs) are now in use worldwide for the treatment of chronic viral hepatitis. Unfortunately, various side effects of IFNs have been reported. Because cytokines, which include IFNs, can affect endocrine function, endocrinological abnormalities are sometimes observed in patients treated with IFNs. We examined the effects of IFN-β on peripheral levels of pituitary and adrenal hormones and cytokines. Six million international units of IFN-β dissolved in glucose solution was injected for 30 min. As a control study, glucose solution without IFN-β was injected. Pituitary hormones (ACTH, GH, TSH, prolactin (PRL), LH, FSH, and arginine-vasopressin (AVP)), cortisol, and cytokines such as interleukin (IL)-1, IL-6, tumor necrosis factor-α (TNF), and interleukin-1 receptor antagonist (IL-1ra) were measured before and after IFN-β injection. The study was carried out on 14 patients with chronic hepatitis type C who were under treatment with IFN-β. All studies were performed when the patients were afebrile. None of the patients had any endocrine or autoimmune diseases. Plasma ACTH levels increased significantly at 60–120 min after IFN-β injection compared with the levels before IFN-β injection and in the control study using glucose injection. Plasma cortisol levels increased after IFN-β injection, in parallel with plasma ACTH elevation. Serum GH levels increased significantly at 120 min after IFN-β injection. All the increased hormones including ACTH, cortisol, and GH, were decreased at the end of the study—180 min after IFN-β injection. Serum levels of TSH, PRL, LH, FSH, and AVP were not changed significantly by IFN-β injection. Plasma IL-1 and TNF levels did not change after IFN-β injection, while IL-6 and IL-1ra were elevated significantly. The increases in IL-6 and IL-1ra were gradual, reaching their peak levels at 180 min after IFN-β injection. However there were no correlations between the hormones measured in this study and the levels of IL-6 or IL-1ra. It would seem that IFN-β has direct or indirect stimulatory effects for ACTH and GH without mediation of the cytokines. These in vivo results are important for investigating the relationship between endocrine and cytokine systems in humans.