The Toxoplasma gondii-specific target antigens for feline immunoglobulin M (IgM) and immunoglobulin G (IgG) immune responses were studied longitudinally using western blot immunoassay in 8 cats experimentally inoculated with T. gondii strain ME49. Multiple antigens were recognized by IgM and IgG during the course of infection. Dense bands were associated with 12 antigens in the IgM western blot immunoassay and 30 antigens in the IgG western blot immunoassay. Immunoglobulin M responses were maximal on week 4 postinoculation (PI) and were greatly diminished by week 20 PI. Immunoglobulin G responses were maximal on week 12 PI. On week 20 PI, the 19-kDa (6/8 samples), 26-kDa (8/8 samples), 28-kDa (8/8 samples), 31-kDa (7/8 samples), 35-kDa (6/8 samples), 51-kDa (6/8 samples), 55-kDa (7/8 samples), and 65-kDa (7/8 samples) antigens were recognized most commonly in the IgG western blot immunoassay. When the western blot immunoassay results were compared to enzyme-linked immunosorbent assay (ELISA) results, there was no clear advantage to the development of IgM-ELISA, IgG-ELISA, IgM western blot immunoassay, or IgG western blot immunoassay using a single antigen instead of multiple antigens as the detection system for the diagnosis of recent infection.