The effect of three soluble stimuli, phorbol myristate acetate, concanavalin A, and the calcium ionophore A23187, on the luminol-enhanced chemiluminescence of human neutrophils was investigated. All three stimuli elicited a strong burst of chemiluminescence which was dose dependent. The effect of phorbol myristate acetate was independent of the presence of divalent cations in the medium and, in fact, was greater in the presence of ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid. The effect of concanavalin A was greatly stimulated by the presence of calcium in the medium, although some reaction was observed in the absence of this cation. In contrast, chemiluminescence induced by A23187 was absolutely dependent upon the presence of extracellular calcium. These results suggest that the mobilization of calcium into the cell is a sufficient, but not necessary, stimulus for initiation of the respiratory burst. Compounds such as phorbol myristate acetate, which act independently of extracellular divalent cations, may function by altering the intracellular ratio of bound/free calcium.