Luminol- and lucigenin-dependent chemiluminescence (CL) was used to compare activation of the respiratory burst of chicken peripheral blood monocytes and heterophils after stimulation with various agents. Monocytes and heterophils were obtained from the blood of three specific-pathogen-free chickens at 14 months of age and purified by a two-step discontinuous Percoll gradient. All cells responded to phorbol 12-myristate 13-acetate (PMA), zymosan A and calcium ionophore A23187 producing CL. The time course of luminol- and lucigenin-dependent CL was similar for both monocytes and heterophils after stimulation with PMA or zymosan A. Heterophils at lower cell number than monocytes responded with similar or higher peak maximum (PM) values. At the concentrations of stimuli used, the order of mean PM values was: zymosan A > PMA > A23187. Addition of 4 x 10(-6) M N-formyl-1-L-methionyl-L-leucyl-L-phenylalanine (fMLP) showed weak but significant CL activity at 1 x 10(6) monocytes per tube with luminol and at 5 x 10(6) monocytes per tube with lucigenin. No significant response to fMLP was observed with heterophils. The results indicate that the respiratory burst of chicken monocytes and heterophils can be measured by CL.