The objective of this study was to compare eight Blastomyces yeast phase lysate antigens, prepared from isolates from northern: N (Minnesota; Wisconsin) and southern: S (Tennessee, Alabama, Mississippi) regions of the United States, for antibody detection in serum specimens from dogs from these same regions with blastomycosis using the indirect enzyme-linked immunosorbent assay (ELISA). In Trial 1, the lysates were evaluated for antibody detection in sera from 36 dogs (N:18; S:18) with the mean absorbance values ranging from 0.672 (N antigen 643) to 1.504 (S antigen T-27). In addition when the N and S sera were evaluated separately, the mean absorbance values ranged from 0.564 (N) to 0.780 (S) with the 643 antigen and from 1.332 (N) to 1.677 (S) with the T-27 antigen. With all eight lysates greater reactivity was evidenced with the S sera than with the N sera. In addition, four combinations of both N and S antigens were compared with regard to antibody detection in the same dog sera as above. Mean absorbance values ranged from 0.967 (N antigen ERC-2 plus S antigen T-27) to 1.355 (N antigen plus S antigen T-27).The greatest reactivity with these combination antigens was less than was determined with the individual S antigen T-27 in Trial 1. In Trial 2 the same 8 lysates were evaluated for antibody detection in sera from 10 immunized rabbits with the mean absorbance values ranging from 0.978 (N antigen 643) to 2.603 (S antigen T-58). Trial 2 also had a total reactivity absorbance value range of 1.503 (N antigens) to 1.870 (S antigens). A higher level of antibody was detected in the serum specimens from infected dogs from S regions and in sera from rabbits immunized with the S isolates than with N dog sera or with N sera from immunized rabbits. Additional studies may provide more evidence with regard to the utilization of N and S lysate antigens for the definitive diagnosis of blastomycosis in animals and humans.