Methotrexate may be an alternative to ciclosporin in the treatment of canine atopic dermatitis (cAD) as suggested by recent data. The aim of the study was to investigate both the tolerance and the pharmacokinetic behavior of methotrexate (MTX) in plasma, following intravenous (i.v.), subcutaneous (s.c.) or oral (OR) administration over several weeks. Six healthy dogs were given oral MTX once a week, respectively, per dog at 2.5 mg/1 week, 5 mg/4 weeks, 7.5 mg/3 weeks, 10 mg/6 weeks and 12.5 mg/5 weeks. No clinically relevant abnormalities of laboratory parameters were noticed. A high inter-individual variation of MTX plasma concentration was observed with a suspicion of saturation phenomenon in absorption. To compare with other routes of administration, six healthy beagle dogs followed a crossover design study at 7.5 mg per dog MTX. The absolute bioavailability was 93% for SC injection and 30% for the oral route. The inter-individual variability was quite low following SC administration compared to oral route. Just as in human, given the substantial variability of oral absorption, clinicians cannot assume consistent oral bioavailability of MTX. Therefore, they may consider switching dogs to the SC route in case of absence of clinical response with a weekly oral dose.