We have examined the ability of mice to survive a lethal challenge with the parasitic hemoflagellate, Trypanosoma cruzi, following immunization with paraflagellar rod proteins (PAR) 1 and 2 either alone or in combination with the following adjuvants: Freund's, alum, QS-21, Ribi-700, or IL-12. PAR administered subcutaneously (sc) in combination with Freund's or alum provided significant protection, 100 and 83%, respectively, against a T. cruzi challenge. In contrast, PAR in combination with QS-21, Ribi-700, IL-12, or Freund's administered intraperitoneally (ip) or PAR alone provide no protection against a challenge. PAR-specific serum antibody titers and isotype profiles for several of the immunization regimens were determined, and no positive correlation could be seen between a protective immune response and either antibody titer or the subclass of antibody induced. We also examined the ability of PAR to stimulate T cells from the spleen and lymph nodes of mice immunized with PAR in combination with Freund's (sc), Freund's (ip), alum, or Ribi-700. Each of the adjuvants strongly enhanced the ability of enriched T cells to proliferate in a PAR-specific fashion, suggesting no obvious correlation between PAR-specific T cell activation and protection. However, examination of the cytokine profiles of the stimulated T cell groups showed that the protective groups differed from the nonprotective groups. While all four groups showed low levels of IL-10, the Freund's (sc) and alum groups had higher levels of IFN-γ and IL-2 than Freund's (ip) and Ribi-700 groups, and most strikingly, no IL-4 could be detected in either the Freund's (sc) or the alum group, in contrast to significant levels of IL-4 in both the Freund's (ip) and the Ribi-700 group. These findings indicate that protective immunity in mice immunized with PAR is associated with a Th1-type response.