The effect of melatonin injection on Freund's adjuvant-induced changes in levels and 24-hr rhythms of circulating ACTH, growth hormone (GH), prolactin (PRL), luteinizing hormone (LH), and insulin was assessed in rats. Animals received subcutaneous (s.c.) injections of melatonin (30 microg) or vehicle, 1 hr before lights off for 12 days. Ten days after melatonin treatment, they were injected with Freund's complete adjuvant or its vehicle s.c., and after 3 days, rats were killed at six different time intervals throughout a 24-hr cycle to measure the different hormones by radioimmunoassay (RIA). Following Freund's adjuvant injection, an increase in serum ACTH, with maintenance of ACTH diurnal rhythm was found. Acrophases of the ACTH rhythm varied from 13:39 to 17:12 hr and the amplitude of rhythm was augmented after immunization. In immunized rats, melatonin treatment increased the amplitude of serum ACTH rhythm. For GH, a depressive effect of immunization on circulating levels, together with absence of diurnal rhythmicity were found. Immunization augmented circulating PRL, while conserving its diurnal rhythmicity. Melatonin-injected rats showed significant diurnal variations of serum PRL after immunization only. Acrophases of the serum PRL rhythm varied from 19:37 to 22:04 hr. Immunization decreased circulating LH and suppressed its 24-hr rhythmicity pattern. The effect of immunization on LH was counteracted by melatonin injection. Acrophases of serum LH rhythm varied from 00:44 to 03:53 hr. Significant effects of immunization and time of day on circulating insulin were detected; immunization increased serum insulin levels with a shift in acrophase from early afternoon to midnight. The data indicate that several early changes in levels and 24-hr rhythms of circulating ACTH, PRL, and LH in Freund's adjuvant-injected rats were sensitive to treatment with pharmacological amounts of melatonin.