Invasion of host cells is essential for the pathogenicity of Salmonella. We have recently shown that invasion of cultured epithelial cells by S. typhimurium is accompanied by activation of the epidermal growth factor receptor. In this report we show that S. typhimurium invasion stimulated a rapid increase in the levels of free intracellular calcium ([Ca2+]i) in cultured epithelial cells. Mutants defective in invasion were unable to induce these calcium fluxes, and addition of calcium antagonists blocked wild-type S. typhimurium entry. These results indicate that [Ca2+]i increase is required for bacterial entry. Further analysis demonstrated that phospholipase A2 and 5-lipoxygenase activities resulting in production of leukotrienes are required for bacterial entry. Addition of the leukotriene D4 to Henle-407 cells caused both an increase in [Ca2+]i and the internalization of an invasion-defective mutant of S. typhimurium. Furthermore, S. typhimurium caused the activation of mitogen-activated protein (also known as extracellular signal-regulated protein) kinase in infected cells.