Using a new enzyme immunoassay (EIA) and standard immunodiffusion (ID) and complement fixation techniques for antibody to the A antigen of Blastomyces dermatitidis, we tested serum from 27 patients with blastomycosis diagnosed histopathologically or by culture; 20 with diagnoses made during 1981 through 1983 (Group A) and 7 during 1974 through 1976 (Group B). We also studied 30 control subjects with Mycoplasma pneumoniae infection (17 subjects), histoplasmosis (6 subjects), coccidioidomycosis (1 subject) and no known disease (6 subjects). Detectable antibody by all 3 tests was present more often for Group A than for Group B, possibly because of delay in testing. Enzyme immunoassay was the most sensitive test; a 1:8 or greater titer was present in acute-phase serum of all Group A patients tested, and a 1:64 or greater titer was associated with disseminated disease (p = 0.003). A positive ID was also more common in disseminated (88%) than in localized (33%) disease. Enzyme immunoassay titers of 1:16 were present in 4 control subjects, 3 with histoplasmosis. The 100% predictive values of a negative EIA and positive ID suggest that these tests are useful for serological screening (EIA) and serologic confirmation (ID) of suspected blastomycosis, particularly in disseminated disease. Enzyme immunoassay titers of 1:32 or greater strongly support the diagnosis and titers of 1:16 or less may indicate localized disease or be nonspecific. None of the serologic tests predicted disease progression or reactivation.