Enzyme immunoassay (EIA), immunodiffusion (ID), and complement fixation (CF) tests for antibody to the A antigen of Blastomyces dermatitidis were assessed in 47 patients in an epidemic of blastomycosis and in 89 control subjects with lower respiratory tract illness. Antibody was detected by EIA, ID, and CF in 77%, 28%, and 9% of the patients, respectively. EIA titers ranged from 1:8-1:512 (median titer, 1:128). Antibody detected by ID or CF was always detectable by EIA. Antibody was detected by EIA 13 days after illness onset, and the peak seroprevalence rate and geometric mean titer occurred 50-70 days after onset. Antifungal therapy produced a significant decline in antibody titer by approximately six months after onset. Seven (8%) control subjects had detectable antibody, six had EIA titers of 1:8, and one had a titer of 1:16. The specificities for EIA, ID, and CF were 92%, 100%, and 100%, respectively. The EIA provides a significant advance in serodiagnostic testing for blastomycosis and can be used in an outbreak setting as an epidemiological tool to identify acute B. dermatitidis infection; titers greater than or equal to 1:32 strongly support the diagnosis, whereas titers of 1:8 or 1:16 are suggestive.