The purpose of this experiment was to elucidate whether the effect of exogenous prolactin (PRL) on immunity parameters of White Leghorn chickens varies during the day. The experiment was carried out on cockerels kept after hatching during 6 weeks under L:D = 12:12 conditions. During 5 consecutive days cockerels were injected with bovine PRL (150 micrograms per bird daily) or its solvent at different time points, i.e., at 0, 4, 8 or 12 HALO (Hours After Light Onset). The birds were sacrificed 24 hours after the last injection at the same time point when the injections were given. It was found that given at 4 HALO prolactin raised, whereas at 8 HALO it decreased the peripheral lymphocyte number. PRL had no effect on granulocyte number, natural anti-rabbit red blood cells (anti-RRBC) agglutinin titre and serum lysozyme activity. Administered at 0 and 12 HALO PRL tended to decrease the thymus and bursa of Fabricius weight. This different PRL effect on peripheral lymphocytes in chickens during the circadian period may be one of the causes of varying effect of this hormone on immunity (stimulatory or suppressive) described in literature. The role of PRL in regulation of immunity is discussed in relation to the possibility of PRL receptors occurrence on chickens lymphocytes.