An enzyme-linked immunosorbent assay for specific antibodies in patients with strongyloidiasis was developed using antigens extracted from the third-stage larvae of Strongyloides stercoralis and Strongyloides ratti. Sera from two groups of patients with strongyloidiasis proven by parasitologic methods, including one group of 29 coded positive sera, and from presumed normal subjects as controls were tested. Sera with optical density values greater than two standard deviations above the mean optical density value for the sera from control subjects were considered to be positive. Using this criterion 82%-84% of 51 positive patients had antibodies to S. stercoralis or S. ratti antigens. Immunosuppression and other factors that might influence antibody responses were associated with some patients who were negative for antibodies to Strongyloides. Antigen from an intestinal nematode of rats, Nippostrongylus brasiliensis, was also evaluated and found to be unsuitable for serodiagnosis of strongyloidiasis because of frequent false-positive as well as false-negative results.