The spleens of horses infected with equine infectious anemia contain an antigen that is useful for a diagnostic immunodiffusion test. This antigen was extracted from the spleen by homogenization of the tissue, centrifugation, and precipitation from the supernatant fluid at 50% saturation with (NH4)2SO4. The antigen was purified by subjecting it to two cycles of electrophoresis in a continuous free-flow electrophoresis cell and finally filtering through a column of Sephadex G-200 gel. The antigen was found to be a small protein with a molecular weight of 27,500 and sedimentation coefficient of 2.1S. Its density was about 1.18 and its isoelectric point 5.8. At 45 to 50 C, it coagulated, losing its antigenicity. The antigen was useful for assaying antibody in the serum from infected horses by using the complement fixation test or the immunodiffusion test. Complement-fixing antibodies were found to be more transient than the precipitating antibodies.