Blastomyces dermatitidis (dog isolate T-58) yeast phase lysate antigen was concentrated and separated by Rotofor preparative isoelectric focusing cell (Bio-Rad). The pH values of the fractions were determined and equilibrated to pH 7.2 and then analysed by enzyme-linked immunosorbent assay using horseradish peroxidase enzyme system against serum specimens from dogs with blastomycosis, histoplasmosis, aspergillosis, and coccidioidomycosis. The results showed a peak absorbance at pH 3.89-4.31 (fractions 4 and 5) with the blastomycosis serum specimens. This was a single sharp peak while the rest of the fractions were lower. In contrast the sera from dogs with histoplasmosis showed a peak absorbance at pH 5.54-5.97 (fractions 9 and 10), while the other mycoses showed patterns that did not resemble the blastomycosis or histoplasmosis specimens. Serum specimens from dogs with blastomycosis being treated with itraconazole were also assayed (pre-treatment and 1, 2, 3, and 12 months post-treatment sera). The characteristic peak for blastomycosis was observed and a decrease in the peak was seen as the treatment progressed. Fractions 3-12 were also used to detect delayed dermal hypersensitivity in hyperimmunized hairless guinea-pigs. Fraction 5 (pH 4.31) elicited the optimal response in B. dermatitidis-immunized animals, while no cross-reactivity was observed in guinea-pigs sensitized with Histoplasma capsulatum killed cells.