Pertussis toxin (PT), a protein toxin of Bordetella pertussis, has many biological activities, including potent adjuvant capacity and the ability to up-regulate immunoglobulin E (IgE) production. Interleukin-4 (IL-4) is a cytokine which is essential for the IgE response. Accordingly, we examined the effect of PT on IL-4 production. Spleen and lymph node cell suspensions were prepared from immunized mice and cultured with antigen or polyclonal stimuli in vitro. IL-4 secretion was assessed by bioassay, and IL-4 mRNA expression was assessed by reverse transcription and polymerase chain reaction. Significantly larger amounts of IL-4 protein and mRNA were produced in vitro by cells from mice given PT at the time of immunization than by cells from mice given antigen alone, PT alone, or the combination of antigen with cyclophosphamide. Total and antigen-specific serum IgE levels were significantly elevated in immunized mice given PT, compared with the other groups. Thus, there was a relationship between serum IgE and IL-4 production. The administration of a single dose of an anti-IL-4 monoclonal antibody in vivo, at the time of immunization and treatment with PT, abolished the development of the IgE response. These results indicate that PT is a potent stimulus for the production of IL-4, which is required for the adjuvant effect of PT on IgE formation.