We have studied the role of protein phosphorylation in the degranulation response of human neutrophils by measuring the effect of ATP depletion and the addition of the protein kinase inhibitor staurosporine in electropermeabilized human neutrophils, activated with Ca2+ and/or GTP-gamma-S. Our studies were carried out in the presence of cytochalasin B to prevent inhibitory effects of actin polymerization on the degranulation response. It was found that protein phosphorylation plays an important role in the degranulation response in cells stimulated with the single stimuli Ca2+ or GTP-gamma-S. However, in neutrophils stimulated with the combination of these activators degranulation can occur without the apparent need for protein phosphorylation, albeit with a slower rate than with protein phosphorylation.