SummaryBordetella pertussis , the etiologic agent of whooping cough, causes disease by employing an array of virulence factors controlled by the BvgA-BvgS twocomponent signal transduction system. Regulation by this system has been extensively characterized in vitro , where bvg -activated genes are repressed in a process known as phenotypic modulation. Differential regulation of these genes by the response regulator BvgA results in promoters that are activated early, middle, or late after being released from modulation. However, the in vivo environmental signal and regulation pattern has not been described. In order to investigate BvgAS-mediated regulation of B. pertussis virulence factors in vivo using the mouse aerosol challenge model, we have adapted the recombinasebased in vivo technology (RIVET) system for use in B. pertussis . We have demonstrated that these strains show resolution during in vitro growth under nonmodulating conditions. In addition, we have demonstrated that modulating strains by growth on media containing MgSO 4 does not affect virulence in the mouse aerosol challenge model. We have therefore used the RIVET system to reveal the time-course of gene expression in vivo for selected B. pertussis virulence factors ( cya , fha , prn and ptx ). Our data indicate that this method can be effectively used to monitor and compare in vivo and in vitro gene expression in B. pertussis , and that temporal regulation patterns previously observed in vitro are mirrored in vivo .