Leukotoxin and endotoxin derived from Pasteurella haemolytica serotype 1 are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Activation of bovine alveolar macrophages with endotoxin or leukotoxin results in the induction of cytokine gene expression, with different kinetics (H. S. Yoo, S. K. Maheswaran, G. Lin, E. L. Townsend, and T. R. Ames, Infect. Immun. 63:381-388, 1995; H. S. Yoo, B. S. Rajagopal, S. K. Maheswaran, and T. R. Ames, Microb. Pathog. 18:237-252, 1995). Furthermore, extracellular Ca²⁺ is required for leukotoxin-induced cytokine gene expression. However, the involvement of Ca²⁺ in endotoxin effects and the precise signaling mechanisms in the regulation of intracellular Ca²⁺ by leukotoxin and endotoxin are not known. In fura-2-acetoxymethyl ester-loaded alveolar macrophages, intracellular Ca²⁺ regulation by leukotoxin and endotoxin was studied by video fluorescence microscopy. Leukotoxin induced a sustained elevation of intracellular Ca²⁺ in a concentration-dependent fashion by influx of extracellular Ca²⁺ through voltage-gated channels. In the presence of fetal bovine serum, endotoxin elevated intracellular Ca²⁺ even in the absence of extracellular Ca²⁺. Leukotoxin-induced intracellular Ca²⁺ elevation was inhibited by pertussis toxin, inhibitors of phospholipases A₂ and C, and the arachidonic acid analog 5,8,11,14-eicosatetraynoic acid. Intracellular Ca²⁺ elevation by endotoxin was inhibited by inhibitors of phospholipase C and protein tyrosine kinase, but not by pertussis toxin, or the arachidonic acid analog. To the best of our knowledge, this is the first report of Ca²⁺ signaling by leukotoxin through a G-protein-coupled mechanism involving activation of phospholipases A₂ and C and release of arachidonic acid in bovine alveolar macrophages. Ca²⁺ signaling by endotoxin, on the other hand, involves activation of phospholipase C and requires tyrosine phosphorylation. The differences in the Ca²⁺ signaling mechanisms may underlie the reported temporal differences in gene expression during leukotoxin and endotoxin activation.