The pathogenic ciliate Ichthyophthirius multifiliis (Fouquet) infects a wide range of freshwater fish and causes the disease Ichthyophthiriasis (Ich) or white spot. Fish that survive infection acquire immunity to subsequent challenge and produce specific serum antibodies that immobilize the parasite in vitro. Cell surface protein antigens targeted by these antibodies are referred to as immobilization antigens (i-antigens). Using immobilizing mouse monoclonal antibodies as ligands, the i-antigens of I. multifiliis were purified by immunoaffinity chromatography. The biochemical characteristics of purified i-antigens were studied with SDS-PAGE, MALDI-TOF, Western blotting, and Edman degradation, and the immunogenicity of purified i-antigens were confirmed by injecting rabbit and channel catfish (Ictalurus punctatus) to produce immobilizing antisera. Subunit vaccines comprised of the purified i-antigens induced protective immunity in juvenile channel catfish when administered with Freunds adjuvant or a CpG oligodeoxynucleotide. Moreover, i-antigen subunit vaccines elicited immobilizing antibodies and conferred protection only against homologous parasites, which supports a model for immunity mediated by immobilizing antibodies. The i-antigen genes were introduced into a novel expression system, the free-living non-pathogenic ciliate Tetrahymena thermophila. The recombinant i-antigens were either targeted to the host cell surface in full-length form or secreted into the culture medium in a truncated form lacking the GPI-anchor addition site. The recombinant i-antigens had the same antigenicity and immunogenicity as i-antigens produced by the parasite. Live cells of Tetrahymena over-expressing Ichthyophthirius i-antigens were effective vaccines against I. multifiliis. DNA vaccines containing the i-antigen genes elicited serum antibody response and conferred protective immunity against I. multifiliis. These results clearly indicate that the i-antigens of I. multifiliis are protective antigens and good candidates for the development of vaccines against I. multifiliis.