Adenylate cyclase toxin (ACT), a virulence factor of Bordetella pertussis, acquires hemolytic and toxic activities after post-translational modification of the cyaA gene product, CyaA. The exact nature of this modification is unknown, but homology to the related repeat toxin alpha-hemolysin of Escherichia coli suggests that fatty acylation of a lysine residue may be involved. In the present study, we used an in vitro chemical approach to acylate unmodified, inactive adenylate cyclase protoxin by using a new water-soluble compound, acylpyrophosphate. We show that undirected transfer of lauric, myristic, or palmitic acid chains to the CyaA protoxin is able to confer both hemolytic and toxic activities to ACT. The chemically modified protoxin shows a specific requirement for Ca2+ ions for toxic activity, as does the wild type toxin. However, the toxic and hemolytic activities of chemically modified ACT are low in comparison to ACT modified in vivo, suggesting that in vitro fatty acylation of the protoxin involves random modification of nucleophilic residues present in the toxin in contrast to the in vivo modification of specific sites.