Staphylococcal α-toxin is a cytolytic toxin secreted by many strains of Staphylococcus aureus that has proinflammatory and cytotoxic effects on human keratinocytes. α-toxin exerts its effects by forming a transmembrane pore that behaves like an ionophore for ions such as calcium. Because cellular membrane disruption with resultant intracellular calcium mobilization is a potent stimulus for the synthesis for the lipid mediator platelet-activating factor, the ability of α-toxin to induce platelet-activating factor production was assessed, and whether the epidermal platelet-activating factor receptor could augment toxin-induced signaling in epithelial cells examined. Treatment of the human keratinocyte-derived cell line HaCaT with α-toxin resulted in significant levels of platelet-activating factor, which were approximately 50% of the levels induced by calcium ionophore A23187. α-toxin also stimulated arachidonic acid release in HaCaT keratinocytes. Pretreatment of HaCaT cells with platelet-activating factor receptor antagonists, or overexpression of the platelet-activating factor metabolizing enzyme acetylhydrolase II blunted α-toxin-induced arachidonic acid release by approximately one-third, suggesting a role for toxin-produced platelet-activating factor in this process. Finally, retroviral-mediated expression of the platelet-activating factor receptor into the platelet-activating factor receptor-negative epithelial cell line KB resulted in an augmentation of α-toxin-mediated intracellular calcium mobilization and arachidonic acid release. These studies suggest that α-toxin-mediated signaling can be augmented via the epidermal platelet-activating factor receptor.