A two-site enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Gnathostoma spinigerum antigens in the sera of parasitized mice. This assay used IgG fractions prepared from serum of a G. spinigerum-infected rabbit as the capture antibody. The same IgG fractions were labeled with alkaline phosphatase and used as an antibody probe. The antigen detection assay was performed along with an antibody detection assay during the course of G. spinigerum infection in mice. Circulating antigen was detected after the first week of infection. The amount of detectable antigen increased steadily until the fourth week, but no significant amount of circulating antigen was detected thereafter. Serum antibody first appeared at the second week. Its level increased steadily until the fourth week, then remained high for at least eight weeks. The sensitivity of the two-site ELISA was approximately 6.75 ng/ml of larval somatic antigen and 27 ng/ml of excretory-secretory antigen. The assay gave false-positive results with Opisthorchis, Trichinella, and Angiostrongylus antigens at the level of 1, 728, 432, and 864 ng/ml or higher, respectively. This antigen detection assay may have application in the diagnosis of human gnathostomiasis.