The production of an antibody which reacts with endocardium, blood vessels, and cardiac interstitial tissue (EVI factor) was experimentally obtained in rabbits immunized either with heterologous heart crude antigen, Trypanosoma cruzi antigen, a mixture of heart and T. cruzi antigens, kidney antigen or lung antigen, and complete Freund's adjuvant. Immunization lasted 2 months, with multiple subcutaneous and foot pad injections being made every 15th day. Preimmunization serum from each animal was used as normal control serum. Rabbit sera were tested by indirect immunofluorescence against mouse heart and skeletal muscle unfixed cryostat sections and revealed by fluoresceinated anti-rabbit globulin diluted 1:80. The highest titers appeared with anti-heart sera (1:192) and with anti-heart/T. cruzi sera (1:96). Incorporation of T. cruzi antigen in heart extract did not increase EVI antibody titers. It is suggested that the EVI factor is an antibody to many antigenic determinants, the majority of which are present in the heart and some of which cross-react with T. cruzi.