Measurements of the level of 1,2-diacylglycerol (1,2-DG) during activation of the respiratory burst of human neutrophils by formyl-methionyl-leucyl-phenylalanine (fMLP) in the presence of platelet-activating factor (PAF) or by opsonized particles show that a correlation between accumulation of 1,2-DG and O2 consumption does not exist. Inhibition of protein kinase C activity with staurosporine before addition of opsonized particles demonstrates that the first phase of the respiratory burst is not inhibited, whereas the second phase, which is accompanied by a rise in the content of 1,2-DG, is strongly inhibited. This study indicates that accumulation of 1,2-DG cannot be the sole signal for the initiation of the respiratory burst in human neutrophils.