We developed an enzyme-linked immunosorbent assay (ELISA) that uses one of two recombinant polypeptides, termed H4/GST and H11/GST, as diagnostic antigens for the detection of antibodies to Toxoplasma gondii in human sera. A total of 59 sera from humans with acute toxoplasmosis, 194 sera from patients with chronic toxoplasmosis, and 151 sera from subjects who were not infected with T. gondii were examined. In all, 68% of the sera from humans with acute toxoplasmosis reacted positively with one or both recombinant T. gondii antigens. By contrast, only 14% of those from patients with chronic toxoplasmosis recognized H4/GST or H11/GST. None of the sera from humans who were not infected with T. gondii, including patients with echinococcosis, entamoebosis, toxocarosis, trichinellosis, glandular fever, or rheumatoid arthritis, recognized H4/GST or H11/GST.