The sensitivity for detection of Histoplasma antigen is lower in serum than in urine. In other antigen assays, treatment of serum at 104 degrees C in the presence of EDTA was required for detection of antigenemia. Sensitivity and specificity for detection of Histoplasma antigenemia were examined with or without EDTA heat treatment of the serum using the MVista Histoplasma antigen enzyme immunoassay. A total of 94.6% of serum specimens from patients with AIDS and histoplasmosis that were negative untreated were positive after EDTA-heat treatment. Two-thirds of the negative serum specimens from patients with probable histoplasmosis, based upon clinical suspicion and Histoplasma antigenuria, were positive after heat treatment. Specificity was 99.0% in controls, including healthy subjects and patients in whom histoplasmosis or blastomycosis, were excluded. Precision and reproducibility were good and excellent, respectively. These findings demonstrate improvement in sensitivity without reduction in specificity, precision, or reproducibility after heat-EDTA treatment.