Anthrax lethal toxin (LT) comprises two proteins: the protective antigen (PA) and the lethal factor (LF). The LT is cytotoxic to macrophage-like cell line J774A.1. Pre-treatment of these cells with neomycin, a phospholipase C inhibitor, protected them against anthrax LT cytotoxicity. Protection obtained with neomycin indicated that LT stimulates phospholipase C in these cells. It was found that levels of inositol 1,4,5-triphosphate (IP₃) dramatically increased in toxin-treated cells. The rise in IP₃ levels was proportional to the dose of LF that was allowed to bind to receptor-bound PA. By using protein kinase C (PKC) inhibitors, we found that the activation of PKC is required for mediating anthrax LT cytotoxicity. Activation of phospholipase C or PKC is not required for the binding of PA to the cell surface receptors or for the uptake or internalisation of the toxin. In this study, we demonstrate that the IP₃ signalling cascade is initiated by anthrax lethal toxin in J774A.1 cells. The second messengers generated during the cascade aid LF in mediating lethality only after its translocation into the cytosol.