Abstract Tacrolimus ointment (TAC) is an effective treatment for atopic dermatitis in humans and dogs. The purposes of the present study were to evaluate the effect of 4 weeks of TAC on intradermal skin testing (IST), and in case of suppression, to investigate if reactivity returned to baseline by 2 or 4 weeks post treatment. Intradermal skin test was performed using saline, histamine, lipopolysaccharide (LPS, 0.4 mg mL−1), house dust (25 PNU mL−1) and house dust mite (1 : 40 000 w/v) at weeks 0, 4, 6 and 8 on nine dogs enrolled in a blinded, crossover, clinical trial, using 0.1% TAC or placebo once daily for 4 weeks. Reactions were evaluated at 15 min, and at 4 and 6 h. Ointment was applied after the 15-min evaluation on weeks 0 and 4. Data were analysed using the statistical software SAS System for Windows. At week 4, TAC did not affect 15-min IST, but some reactions in the TAC group were suppressed at 6 h compared to baseline. In the TAC group, 4-h IST reactivity was reduced 2 weeks after discontinuation but returned to baseline by 4 weeks. In conclusion, TAC has no effect on immediate reactions but decreased some late-phase reactions. Therefore, no withdrawal is recommended to evaluate only immediate reactions, but a 4-week withdrawal may be necessary for evaluation of late-phase reactions.