We have studied the effects on interferon-gamma (IFN-gamma) production of pertussigen, a protein toxin from Bordetella pertussis that augments and prolongs delayed-type hypersensitivity (DTH) reactions. Lymphoid cell suspensions from immunized mice were incubated with antigen or mitogen, and the culture supernatants were assayed for IFN-gamma. The production of IFN-gamma on exposure to specific antigen or concanavalin A was greatly enhanced if mice were given pertussigen at the time of immunization. There was no detectable IFN-gamma production when cells were exposed to saline or to an irrelevant antigen. The effect of pertussigen on antigen-driven IFN-gamma production correlated with its effect on the capacity of the same cell populations to transfer DTH. The enhanced IFN-gamma production by cells from mice given pertussigen could not be attributed to an increased antigen-presenting capacity of this cell population. Production of IFN-gamma was abolished if the cells were pretreated with emetine, but not with mitomycin C, and the release of IFN-gamma was not detected in the first 8 hr of culture. After immunization with pertussigen, IFN-gamma was produced by lymph node and spleen cells from 7 days onward and both cell types produced IFN-gamma until at least 30 days after immunization. It is suggested that the augmentation of antigen-specific IFN-gamma production may contribute to the prolonged DTH reactions induced by pertussigen in vivo.