The interaction of Listeria monocytogenes with endothelial cells represents a crucial step in the pathogenesis of listeriosis. Incubation of human umbilical vein endothelial cells (HUVEC) with wild-type L. monocytogenes (EGD) provoked immediate strong NO synthesis, attributable to listerial presentation of listeriolysin O (LLO), as the NO release was missed upon employment of a deletion mutant for LLO (EGD hly mutant) and was reproduced by purified LLO. Studies of conditions lacking extracellular Ca²⁺ suggested LLO-elicited Ca²⁺ flux as the underlying mechanism. In addition, HUVEC incubation with EGD turned out to be a potent stimulus for sustained (>12-h) upregulation of proinflammatory cytokine generation (interleukin 6 [IL-6], IL-8, and granulocyte-macrophage colony-stimulating factor). Use of deletion mutants for LLO (EGD hly mutant), listerial phosphatidylinositol-specific phospholipase C (EGD plcAmutant), broad-spectrum phospholipase C (EGD plcB mutant) and internalin B (EGD inlB mutant), as well as purified LLO, identified LLO as largely responsible for the cytokine response. Endothelial cells responded with diacylglycerole and ceramide generation as well as nuclear translocation of NF-κB to the stimulation with the LLO-producing strains EGD andListeria innocua. The endothelial PC-phospholipase C inhibitor tricyclodecan-9-yl-xanthogenate as well as two independent inhibitors of NF-κB activation, pyrolidine dithiocarbamate and caffeic acid phenethyl ester, suppressed both the NF-κB translocation and the upregulation of cytokine synthesis. We conclude thatL. monocytogenes is a potent stimulus of NO release and sustained upregulation of proinflammatory cytokine synthesis in human endothelial cells, both events being largely attributable to LLO presentation. LLO-induced transmembrane Ca²⁺ flux as well as a sequence of endothelial phospholipase activation and the appearance of diacylglycerole, ceramide, and NF-κB are suggested as underlying host signaling events. These endothelial responses to L. monocytogenes may well contribute to the pathogenic sequelae in severe listerial infection and sepsis.