A number of bacterial protein toxins, including adenylate cyclase (AC) toxin from Bordetella pertussis, require the product of an accessory gene in order to express their biological activities. In this study, mass spectrometry was used to demonstrate that activated, wild-type AC toxin was modified by amide-linked palmitoylation on the epsilon-amino group of lysine 983. This modification was absent from a mutant in which the accessory gene had been disrupted. A synthetic palmitoylated peptide corresponding to the tryptic fragment (glutamine 972 to arginine 984) that contained the acylation blocked AC toxin-induced accumulation of adenosine 3’,5’-monophosphate, whereas the nonacylated peptide had no effect.