Comparative evaluations were performed to assess the stability, sensitivity and specificity of eight lots of yeast lysate antigen prepared from a Blastomyces dermatitidis dog isolate (T-58). These antigens were prepared during the period from 1989 to 1995. The lysates were used in an ELISA for the detection of antibodies in serum specimens from dogs with blastomycosis and histoplasmosis. In order to evaluate the ability of the lysates to elicit delayed dermal hypersensitivity (DTH) responses, they were compared as skin-testing antigens in guinea pigs that were previously sensitized with B. dermatitidis or Histoplasma capsulatum killed whole yeast cells. All 8 of the lots of antigen detected antibody in the sera from dogs with blastomycosis (absorbance values ranged from 0.432 to 0.543; mean value of 0.508). The absorbance values ranged from 0.283 to 0.439 (mean value of 0.326) when the lysates were assayed against sera from dogs with histoplasmosis. All of the antigens were able to elicit a DTH response in B. dermatitidis immunized animals (mean axes of induration values ranged from 10.5 mm to 12.5 mm; mean value of 11.6 mm). In contrast, only minimal cross-reactivity was evidenced in the guinea pigs immunized with H. capsulatum (mean axes of induration values ranged from 0 to 4.5 mm; mean value of induration of 1.7 mm).