The second-generation MVista Blastomyces antigen enzyme immunoassay was not quantitative; therefore, specimens obtained previously were tested in the same assay as new specimens to assess the change in antigen levels. Furthermore, the sensitivity in serum had not been fully evaluated. The purpose of this study was to evaluate a quantitative Blastomyces antigen assay and detection of antigen in serum. Calibrators containing known concentrations of Blastomyces galactomannan were used to quantify antigen in urine and serum from patients with proven blastomycosis and from controls. Paired current and previously obtained urine specimens were tested to determine if quantification eliminated the need for concurrent testing to assess change in antigen. Pretreatment of serum with EDTA at 104°C was evaluated to determine if dissociation of immune complexes improved detection of antigenemia. Antigenuria was detected in 89.9% of patients with culture- or histopathology-proven blastomycosis. Specificity was 99.0% in patients with nonfungal infections and healthy subjects, but cross-reactions occurred in 95.6% of patients with histoplasmosis. Change in antigen level categorized as increase, no change, or decrease based on antigen units determined in the same assay agreed closely with the category of change in ng/ml determined from different assays. Pretreatment increased the sensitivity of detection of antigenemia from 35.7% to 57.1%. Quantification eliminated the need for concurrent testing of current and previously obtained specimens for assessment of changes in antigen concentration. Pretreatment increased the sensitivity for detection of antigenemia. Differentiation of histoplasmosis and blastomycosis is not possible by antigen detection.