Four recombinant forms of the cell-invasive adenylate cyclase toxin (CyaA) of Bordetella pertussis were compared for the ability to enhance protection against B. pertussis in mice when coadministered with an acellular pertussis vaccine (ACV). The four forms were as follows: fully functional CyaA, a CyaA form lacking adenylate cyclase enzymatic activity (CyaA*), and the nonacylated forms of these toxins, i.e., proCyaA and proCyaA*, respectively. None of these forms alone conferred significant (P > 0.05) protection against B. pertussis in a murine intranasal challenge model. Mice immunized with ACV alone showed significant (P < 0.05) reductions in bacterial numbers in the lungs after intranasal challenge compared with those for control mice. When administered with ACV, both CyaA and CyaA* further reduced bacterial numbers in the lungs of mice after intranasal challenge compared with those for ACV-immunized mice, but the enhanced protection was only significant (P < 0.05) with CyaA*. Coadministration of CyaA* with ACV caused a significant (P < 0.05) increase in immunoglobulin G2a antibody levels against pertactin compared with those in mice immunized with ACV alone. Spleen cells from mice immunized with ACV plus CyaA* secreted larger amounts of interleukin-5 (IL-5), IL-6, gamma interferon (IFN-gamma), and granulocyte-macrophage colony-stimulating factor (GM-CSF) than did cells from mice immunized with ACV plus CyaA or ACV alone after stimulation in vitro with a mixture of B. pertussis antigens. Spleen cells from mice immunized with ACV plus CyaA* also secreted larger amounts of IFN-gamma and GM-CSF than did cells from mice immunized with CyaA* alone after stimulation in vitro with CyaA*. Macrophages from mice immunized with ACV plus CyaA* produced significantly (P < 0.05) higher levels of nitric oxide than did macrophages from mice immunized with CyaA* alone, ACV alone, or ACV plus CyaA after stimulation in vitro with a mixture of B. pertussis antigens or heat-killed B. pertussis cells. These data suggest that the enhancement of protection provided by CyaA* was due to an augmentation of both Th1 and Th2 immune responses to B. pertussis antigens.