In order to study the signal transduction mechanism of human endothelial cells (EC), the regulation of superoxide anion (O₂⁻) release in EC has been investigated using the calcium ionophore A23187 and phorbol myristate acetate (PMA), a potential activator of the Ca²⁺ activated, phospholipid‐dependent protein kinase, designated “protein kinase C.” PMA enhanced O₂⁻ release from EC, and this enhancement occurred regardless of the presence or absence of extracellular Ca²⁺. A similar increase was produced by A23187; omission of extracellular Ca²⁺ prevented this increase. Simultaneous stimulation with PMA and A23187 produced a large increase in O₂⁻ release at submaximal concentrations of these agents, which, when added separately, caused minimal effects. These findings indicate that the activation of protein kinase C and mobilization of Ca²⁺ evoked by PMA and A23187 respectively are synergistically effective for eliciting a full physiological response of EC in the generation and release of O₂⁻.