Antisera to acetone-fixed tachyzoites were prepared by immunizing rabbits intravenously with the fixed organisms. Immunoblots in which purified immunoglobulin G (IgG) of the antisera was used recognized four antigens of a supernatant (after centrifugation at 10,000 x g for 30 min) fraction of sonicated tachyzoites. A purified toxoplasma antigen fraction, referred to as acute-stage-specific (AC) antigen, that contained mainly three antigens was obtained by affinity chromatography by using the purified IgG. One antigen had a molecular weight of approximately 52,000, and two antigens had molecular weights of approximately 6,000. An enzyme-linked immunosorbent assay (AC-ELISA) in which this purified antigen fraction, obtained by affinity chromatography, was used for detection of IgG antibody detected early acute infection in sera of patients with toxoplasmic lymphadenopathy. Ninety-two percent of sera obtained within 2 months after onset of lymphadenopathy were positive in the AC-ELISA, whereas only 9% of sera obtained later than 5 months after onset of the clinical illness were positive. The AC-ELISA appears highly sensitive and specific for diagnosis of acute toxoplasmosis.