The origin of chemiluminescence in human granulocytes was studied by comparing stimulation of chemiluminescence by the calcium ionophore and nonreceptor stimulant, A23187, to that of the known receptor stimulant, formylmethionyl-leucyl-phenylalanine (FMLP). Granulocytes isolated from human blood were partially purified by dextran settling and analyzed for chemiluminescence, oxygen consumption, superoxide release, hydrogen-peroxide release, and membrane potential. A23187 induced chemiluminescence was inhibited by the calmodulin inhibitor, R24571, and the antiinflammatory drug, MK830. The potency of nonsteroidal drug effects on A23187 induced chemiluminescence, ranked from greatest to least inhibition, was BW755C, sulindac-sulfide, butazolidine, sulindac-sulfone, and indomethacin. The data indicated that inhibition of the cyclooxygenase pathway alone was less effective than inhibition of both the lipoxygenase and cyclooxygenase pathways of arachidonic- acid metabolism in blocking A23187 mediated chemiluminescence. The phospholipase-A2 inhibitor, quinacrine, completely blocked A23187 induced chemiluminescence but only partially blocked FMLP stimulated chemiluminescence. Oxygen consumption by human granulocytes was not enhanced by the addition of A23187 to the medium but was significantly increased by subsequent treatment with FMLP. The sequential addition of A23187 and FMLP also produced two separate depolarizations of the membrane with two sequential chemiluminescent peaks. The authors conclude that A23187 chemiluminescence is dependent on calmodulin, independent of the oxidative burst, and attributable to arachidonate release from cellular phospholipids.