We have previously shown that both atopic and normal dogs generate an IgG response to antigens of Malassezia pachydermatis. The aim of this study was to compare IgE responses to separated proteins of M. pachydermatis in 28 atopic dogs with Malassezia dermatitis and 22 clinically normal dogs using Western immunoblotting. Six different detection systems were evaluated in order to assess sensitivity and eliminate nonspecific binding and cross-reactivity. The protocol yielding the best results utilized a monoclonal mouse antidog IgE, an alkaline phosphatase conjugated goat antimouse IgG which had been passed through a canine IgG column 3 times, a chemiluminescent substrate and a digital imaging system. Proteins of 45, 52, 56 and 63 kDa were recognized by more than 50% of the atopic dog sera and thus represented major allergens. Only a minority of normal dogs showed faint IgE binding to these proteins. The results indicate that the majority of atopic dogs with Malassezia dermatitis have a greater IgE response than normal dogs, suggesting an IgE-mediated immune response may be clinically important in the pathogenesis of the disease.