An enzyme-linked immunosorbent assay (ELISA) for the detection of specific IgG and IgM antibody to Pseudomonas pseudomallei was developed. The IgG-ELISA was compared with the indirect fluorescence assay for IgG antibody (IgG-IFA) and the indirect hemagglutination (IHA) test in studies with serum specimens from persons from endemic areas for melioidosis and from persons from nonendemic areas of Australia. The sensitivity and specificity of the IgG-ELISA were 90% and 99%, respectively, comparable to those obtained with the IgG-IFA. The IgG-ELISA was more sensitive than the IHA test (74%) and was more suitable than the IgG-IFA as a serologic screening test for melioidosis. The IgM-ELISA was compared with the IgM-IFA as a marker of disease stage in patients with melioidosis. There was good diagnostic agreement between the tests; 92% of patients with active disease gave IgM-ELISA titers greater than or equal to 1:5,120 and 93% of patients with subclinical melioidosis had IgM-ELISA titers less than or equal to 1:1,280. Of the overlap group of patients with a borderline IgM-ELISA titer of 1:2,560, approximately 33% were clinical cases. An uncommon disease stage consisting of a self-limited, short-term, flu-like, pyrexial illness accompanied by elevated serum IgM-ELISA titers (greater than or equal to 1:5,120) was seen in a small number of patients residing in endemic Australia.