Abstract The purpose of this study was to evaluate reactions to intradermal injections of Tyrophagus putrescentiae extract in healthy dogs and dogs with atopic dermatitis and to compare the prevalence of positive reactions in the two groups. Twenty-one healthy dogs and 26 atopic dogs were tested intradermally with T. putrescentiae extract at 1000, 500, 250, 125, 63, 32 and 16 PNU/mL. Reactions were evaluated objectively and subjectively. A Mann–Whitney test was used to determine differences in grade of reaction to storage mites between healthy dogs and dogs with atopic dermatitis. Positive reactions to storage mite extract were most common at 1000 PNU/mL with approximately one third of normal and atopic dogs showing a positive reaction to T. putrescentiae. There was no significant difference in the incidence of positive reactions between normal and atopic dogs for any of the Tyrophagus extract concentrations.