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Pasteurella multocida produces a 146-kDa protein toxin (PMT), which activates multiple cellular signal-transduction pathways, resulting in the activation of PLCbeta, Rho, JNK, and ERK. In addition to an essential cysteine residue at position 1165, PMT contains several histidine residues in the catalytically important C-terminal part of the protein. To elucidate the role of the histidine residues, we treated PMT with the histidine-modifying substance diethyl pyrocarbonate (DEPC). DEPC inhibited PMT in a time- and concentration-dependent manner, suggesting that one or several histidine residues are essential for the biological activity of PMT. In experiments in which PMT was directly delivered into the cytosol of EBL cells by electroporation, we show that DEPC treatment inhibits the catalytically important histidine residues. Leucine substitutions of eight individual histidine residues in the C-terminal catalytic domain of PMT were constructed, and the effect on the biological activity of PMT was analyzed by determining PLCbeta, Rho, and ERK activation. Substitution of two histidine residues, H1205 and H1223, led to inactivation of the resulting PMT proteins, indicating that H1205 and H1223 play an important role in biological activity of the toxin. In addition, we show that the mutant toxins appear to be correctly folded, as judged by protease digestion. The precise function of H1205 and H1223 is not yet known. However, treatment of PMT with the cation chelating substance 1,10-phenantroline led to inactivation of the toxin, indicating that the essential histidine residues and cysteine 1165 might be involved in metal ion binding.