Pertussis toxin and adenylate cyclase toxin both contribute to the pathogenesis of whooping cough. Production of these proteins is controlled by the bvg locus, which is inactive at 25 degrees C, but at 37 degrees C produces a Vir+ phenotype. In view of the temperature dependence of virulence factor synthesis, the effects of temperature and host factors on their action were examined. The NAD glycohydrolase activity of the S1 subunit of pertussis toxin was enhanced by CHAPS, a zwitterionic detergent, with a temperature optimum of approximately 35 degrees C. Similar temperature optima for the ADP-ribosylation by pertussis toxin of transducin and recombinant Go alpha were observed. Since the temperature--activity relationship of S1 differed from that of S1 in activated holotoxin, and since S1 in activated holotoxin was more stable at 42 degrees C than was S1, it appears that S1 associated with the B oligomer components may, in fact, be an active species. Bordetella pertussis adenylate cyclase is activated by a host factor, calmodulin. In the absence of calmodulin, the temperature optimum for enzymatic activity was approximately 25 degrees C, whereas in its presence it was approximately 35 degrees C. Thus, the temperature optima for pertussis and adenylate cyclase toxins, virulence factors whose production is increased through the bvg locus at physiological temperatures, are either at or near these temperatures when stimulated by host factors.