This study evaluated the effect of insulin on the respiratory burst of human polymorphonuclear leukocytes (PMNLs) and the signalling pathways involved in this process, especially the involvement of protein kinase C (PKC). Isolated human PMNLs from healthy volunteers were incubated with different concentrations of insulin (10<sup>-10</sup>-10<sup>-7</sup> mol/l) and for different durations of incubation (5-90 min). The intracellular production of hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) was detected employing a previously validated flow cytometric assay using 2’,7’-dichlorofluorescein-diacetate (DCFH-DA) as a marker for H<sub>2</sub>O<sub>2 </sub>production. Specificity of insulin action was verified using an insulin antagonist (the monoclonal antibody MA-10). To identify the signalling pathway involved, we used: (a) monoclonal antibody MA-5, directed against the α-subunit of the insulin receptor, that partially mimics insulin without activating tyrosine kinase; (b) H7, an inhibitor of PKC involved in O<sub>2</sub><sup>-</sup> production in PMNLs, and (c) phorbol myristate acetate (PMA) that binds and stimulates PKC. Insulin caused a dose- and time-dependent stimulation of H<sub>2</sub>O<sub>2</sub> release by human PMNLs. The effect of insulin was blocked by MA-10. The actions of insulin and PMA on H<sub>2</sub>O<sub>2</sub> release were additive, whereas the actions of MA-5 and PMA were not. H7 partially inhibited the H<sub>2</sub>O<sub>2</sub> production stimulated by insulin and completely inhibited MA-5 action. We conclude that insulin stimulates, in a dose- and time-related manner, the respiratory burst of human PMNLs. PKC activation can only partially account for the intracellular mechanisms involved in this process.