BACKGROUND Atopic dogs with hypersensitivity to Dermatophagoides farinae (Df) have IgE recognizing high molecular weight (MW) allergens more often than the low MW Der f 1 and 2. A new high MW Df allergen, Zen-1, has been identified recently. OBJECTIVES To determine the IgE reactivity of American and European Df-hypersensitive dogs to Zen-1, Der f 1 and Der f 2. METHODS We tested sera from 33 Df-reactive dogs from the USA, 29 from Europe and 15 experimentally sensitized to Df, by ELISA against crude Df, Der f 1, Der f 2 and Zen-1. ELISA inhibition was performed with sera reactive to Zen-1. Intradermal testing (IDT) was also done with the same allergens in 25 other American atopic dogs. RESULTS Altogether, IgE seropositivity to Zen-1 was more prevalent (86%) than that to Der f 1 (17%) or Der f 2 (19%). The IgE reactivity to Zen-1 was correlated to that against crude Df; this allergen alone inhibited a high percentage (median: 50%; range: 22-84%) of the binding to the crude mite extract. The seropositivity to low MW allergens was highest in experimentally sensitized dogs. Serum IgE recognition of Der f 1 was low in dogs with AD; that to Der f 2 was significantly lower in American dogs (6%) than in European ones (28%). A high prevalence of positive immediate IDT reactions to Zen-1 confirmed the likely relevance of serological results. CONCLUSIONS AND CLINICAL IMPORTANCE This study establishes Zen-1 as a major allergen in atopic dogs sensitized to Df.