The effect of pinealectomy and melatonin injections on the diurnal rhythms of serum lysozyme and blood granulocytes was examined in White Leghorn cockerels kept from time of hatching for 5 weeks in L:D 12:12 conditions and immunized twice with sheep red blood cells (SRBC). Pinealectomy or sham-operation was made during first week of life. Pinealectomized chickens were injected daily with a melatonin dosage increased over 4 consecutive weeks (the dosage was 10, 13, 16, and 20 ng per bird daily during the 4 weeks, respectively; MEL I) at the beginning of darkness. The same treatment was performed on chickens with an intact pineal gland using additional melatonin doses increased 10 times (MEL II) and 500 times (MEL III). Intact chickens were also injected with MEL II and MEL III 4 hr before the end of light. Control birds received equivalent injections of vehicle. Five-week-old chickens were sacrificed during a 24-hr period every 4 hr. The existence of diurnal rhythm was evaluated by cosinor analysis. Pinealectomy shifted the acrophase of the diurnal rhythm of granulocytes and abolished that of serum lysozyme. Both rhythms were restored in pinealectomized chickens by MEL I but not by vehicle injections. The same melatonin dose was unable to change the granulocyte rhythm but delayed the acrophase of that of serum lysozyme in chickens with an intact pineal gland. Two higher melatonin doses influenced the diurnal rhythm of granulocytes as a function of dose and time of administration. The rhythm of serum lysozyme was dependent only on the time of injection. The pineal gland seems to control, via its hormone melatonin, the diurnal rhythm of nonspecific immunity in chickens.