Human as well as canine and rat polymorphonuclear cells (PMN) were separated from whole blood by centrifugation. Two-step discontinuous Percoll gradients with distinct different densities were used. The chemiluminescence properties of the isolated P M N and of phagocytes in small quantities of whole blood were compared in luminol-enhanced assays after stimulation with various agents: non-opsonized zymosan (3.5 g/l), phorbol myristate acetate (PMA, 2.8 X lop6 mol/l), calcium ionophore A 23187 mol/l) and N-formylmethionyl-leucyl-phenylalanine (FMLP, 3.5 X mol/l). The isolated cells of the three species responded t o all of the various stimuli. Species-related sensitivity could be ordered: human > canine > rat. Response t o the various agents in the human cells can be ranked: PMA 3 A 23187 > zymosan > FMLP; for the dog: A 23187 > PMA > zymosan > FMLP; and for the rat: zymosan 5 PMA > FMLP 3 A 23187. Time course and peak maximum response were different upon stimulation in the absence and presence of autologous plasma. Distinct soluble stimuli resulted in maximum responses below the baseline in the whole blood assays with canine (FMLP) and rat (FMLP, A 23187) phagocytes.