This paper describes a new procedure of preparation of the antigen for an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against Toxoplasma gondii. To examine the reliability of this ELISA using whole trophozoites produced in a serum-free tissue culture as an antigen, 221 sera were tested comparatively in the new system (TTE, total trophozoites ELISA), in the indirect fluorescent antibody test (IFAT), and in a commercially available ELISA using sonicated trophozoites as an antigen (STE, sonicated trophozoites ELISA). The ELISA with antigen lysate showed a good correlation with the IFAT; however, false-negative results were sometimes obtained. The TTE was performed with all sera in two modifications: one test with an anti-IgG conjugate (G-TTE) and the other with an anti-Ig-G, -M, -A conjugate (GMA-TTE). In none of these TTE modifications were insensitivities observed; however, the G-TTE seems to offer a clearer differentiation between specifically reactive and nonreactive findings. The present study shows that the ELISA with whole trophozoites produced in serum-free tissue culture might be used as an alternative test to the IFAT. This test combines the advantages of the ELISA system with the sensitivity and specificity of the IFAT.