BALB/c mice were immunized subcutaneously with soluble Neospora caninum tachyzoite antigen (NSO) entrapped in nonionic surfactant vesicles (NISVs) or administered with Freund's complete adjuvant (FCA). Following virulent parasite challenge, groups of mice immunized with NSO and either NISVs or FCA had clinical neurological disease and increased numbers of brain lesions compared to groups of mice inoculated with FCA, NISVs, or phosphate-buffered saline (PBS) alone. Increased numbers of brain lesions were statistically significant only between mice immunized with NISV-NSO and NISV- or PBS-treated mice. Following parasite challenge, brain inflammatory infiltrates in all experimental and control groups of mice were relatively similar and consisted of compact infiltrates of macrophages admixed with various numbers of lymphoid cells. Increased brain lesions in NSO-immunized mice were associated with increased antigen-specific interleukin 4 (IL-4) secretion and increased IL-4:gamma interferon secretion ratios from splenocytes in vitro and increased antigen-specific immunoglobulin G1 (IgG1):IgG2a ratios in vivo. Thus, immunization with whole killed N. caninum antigen and either liposoidal or Freund's adjuvant induced a type 2 immune response that was associated with worsened disease. The present studies emphasize the need to identify specific N. caninum antigens or other delivery systems that will elicit protective immune responses to neosporosis.