The purpose of this study was to investigate the immunoregulatory potential of Hsp60 in the skin of dogs with atopic dermatitis. Three dogs with chronic atopic dermatitis and four healthy dogs were injected intradermally with Hsp60 and phosphate-buffered saline. Biopsies were taken before testing from non-injected control skin, lesional and non-lesional atopic skin, and 48 and 72 h after injection. Analysis of cytokine messenger RNA was performed using quantitative real-time polymerase chain reaction. Forty-eight hours after Hsp60 injection, a rise in interleukin (IL)-10 was found (P = 0.034) with the highest expression levels in non-lesional atopic and control skin. A rise of transforming growth factor beta (P = 0.015) and IL-12p40 (P = 0.017) was noticed 72 h after Hsp60 injection in control skin. No significant differences were observed for the expression of IL-4, IL-12p35, and interferon gamma. The results indicate that Hsp60 is able to induce cytokines of a regulatory and Th1 phenotype in the skin. Furthermore, this study seems to provide a first indication of deficient Hsp60 response in atopic dermatitis affected skin.