This study aimed to investigate both the pharmacokinetic behavior and tolerance of methotrexate (MTX) in horses to design a specific dosing regimen as a new immunomodulatory drug for long-term treatment. To determine the primary plasma pharmacokinetic variables after single intravenous, subcutaneous or oral administration, six horses were administered 0.3 mg/kg MTX in a crossover design study. After a 10-week washout, MTX was administered subcutaneously to three of the six previously treated horses at a dose of 0.3 mg/kg once per week for 3 months. In both studies, MTX and metabolite concentrations were measured using LC-MS/MS. The absolute bioavailability of MTX was 73% following subcutaneous administration but less than 1% following oral administration. The plasma clearance was 1.54 ml min-1 kg-1 (extraction ratio = 2%). After 24 hr, plasma concentrations were below the LOQ. No adverse effects were noted except for a moderate reversible elevation in liver enzymes (GLDH). With regards to the main metabolites of MTX, very low concentrations of 7-hydroxy-MTX were found, whereas polyglutamated forms (mainly short chains) were found in red blood cells. A subcutaneous dose of 0.2 mg kg-1 week-1 may be safe and relevant in horses, although this has yet to be clinically confirmed.