To evaluate the effects of methotrexate (MTX) on the biology of a vascularized bone graft (VBG), a study of adult dogs was performed. Different protocols of treatment using MTX, with or without the administration of citrovorum rescue factor (CRF), were studied. A controlled experiment involving 18 dogs was performed to evaluate the biology of a free vascularized rib graft used to reconstruct a 7-cm segmental defect in the femoral shaft. The grafts were examined at five, eight, and 14 weeks after surgery. Bone union, remodeling, and vasculature were found to be normal, despite a high rate of cortical necrosis in the early follow-up periods (3040%). In a similar group of VBG, MTX was administered at a dose of 1500 mg/m2 body surface (intravenous) from the 14th postoperative day, every three weeks, until the animals were killed. Citrovorum rescue factor was administered to avoid the toxic side effects of the MTX. The results of this group did not differ from those of the control group as far as bone union, remodeling, vasculature, and viability of the graft were concerned. Methotrexate did not, for the most part, affect the biology of a free vascularized bone graft.