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To investigate IgG responses to Dermatophagoides farinae antigens, a semi-quantitative, Western blot, digital image analysis system was developed and validated. Both healthy and atopic dogs mounted D. farinae-specific total IgG, IgG1 and IgG4 responses to separated antigens. D. farinae-specific IgG2 and IgG3 responses were difficult to detect. The most commonly recognised band in both groups was a 98 kDa antigen, most likely to be the major allergen Der f 15. These results indicate that D. farinae antigens are recognised by the canine immune system regardless of whether or not a dog is atopic. They also demonstrate that antibody class switching to IgE in atopic dogs does not appear to inhibit IgG production. In atopic dogs undergoing allergen-specific immunotherapy (ASIT) against D. farinae with alum-precipitated vaccines, there was no significant increase in D. farinae-specific total IgG, IgG1 or IgG4, even in dogs showing apparent clinical improvements. In contrast, ASIT using aqueous vaccines resulted in significant increases in D. farinae-specific IgG responses. These results suggest that aqueous ASIT may elicit the production of IgG blocking antibodies in atopic dogs, an effect not detected using alum-precipitated vaccines. To investigate the cytokine milieu in dogs with atopic dermatitis, real-time quantitative RT-PCR was used to detect the expression of mRNA transcripts of the Th1 cytokine IFN-γ, the Th2 cytokine IL-4, the Treg cytokine TGF-β and inducible NO synthetase (iNOS) as a measure of the innate immune response. The expression of TGF-β and iNOS were significantly lower in lesional skin compared to non-lesioned skin in atopic dogs, whilst IFN-γ was expressed in a significantly lower level in lesional skin compared to healthy controls. IL-4 expression did not differ between the groups.