The effect of daily injection of the pineal hormone melatonin and naltrexone, an opioid antagonist, on the circadian rhythm and the level of immune parameters (plaque forming cell [PFC] number, serum agglutinin titer, lymphoid gland weight, total white blood cells (WBC) and their fraction number, and serum lysozyme [LZ] content) was examined in White Leghorn cockerels and female BALB/c mice kept in LD 12:12. Animals were immunized ip with sheep red blood cells (SRBC) to stimulate their immune system. Subcutaneous injections of melatonin, naltrexone, or both drugs together were made 2 hr before the end of light, for 4 or 5 days, beginning on the day of immunization. The day following the fifth injection, chickens were sacrificed over a 24 hr period every 4 hr (experiment I) or twice daily, i.e., at the beginning of light and dark phases (experiment II). Mice were killed on the day following the fourth injection at the beginning of light, and splenic PFC number was determined (experiment III). In experiment I, the existence of the diurnal rhythm was evaluated by cosinor analysis. Melatonin injections entrained the circadian rhythm in anti-SRBC serum agglutinins, but it did not influence circadian rhythmicity in other parameters examined. The circadian rhythm in total WBC number and their fractions was entrained by naltrexone treatment. Melatonin injections did not affect either the diurnal mean of parameters examined or the weight of lymphoid organs. Splenic PFC number in chickens was diminished by both melatonin and naltrexone injections, whereas in mice it was increased by melatonin, and naltrexone antagonized that effect.(ABSTRACT TRUNCATED AT 250 WORDS)