Transport of proteins across cellular membranes is a central process in cell physiology. Many protein toxins from poisonous plants or pathogenic bacteria are able to penetrate into the cytosol of their target cells where they exert their toxic effects. Cell membrane trafficking of proteins to reach the cytosol is referred to as translocation. Here, we propose to decipher the membrane insertion process of the adenylate cyclase (CyaA) toxin produced by Bordetella pertussis, the causative agent of whooping cough. We propose to study the full-length CyaA toxin, as recently, we are able to produce a monomeric, stable, folded and functional toxin in physiological buffers. Our objective is to describe the translocation process of CyaA to help us to improve the design of therapeutic strategies against Pertussis and our biotechnological applications.