Methotrexate (MTX) is lethal or teratogenic to embryos of all species tested. New Zealand white rabbit embryos are relatively resistant to the embryolethal effects of MTX. However, when pregnant does were injected iv with 19.2 mg MTX/kg on gestational day 12, virtually all surviving fetuses exhibited multiple malformations of the head, limbs, and trunk. MTX is a structural analogue of folic acid that competitively inhibits dihydrofolate reductase, thereby preventing formation of folinic acid and essentially stopping one carbon metabolism. One carbon metabolism is important in the synthesis of methionine, histidine, glycine, and purine bases that are required for the de novo synthesis of DNA. Presumably these metabolic effects of MTX relate directly to its mechanism of developmental toxicity. An ameliorative treatment has been tested utilizing iv injection of pregnant rabbits with leucovorin (LV), a close structural analogue of folinic acid (the product of the inhibited enzyme), at various times after MTX exposure. When LV was injected at times up to 24 hours after MTX, fewer malformed fetuses resulted and the incidence of specific malformations was reduced. When given at times up to 20 hours after MTX administration, LV virtually eliminated the grossly apparent effects of MTX at term. In the forelimb bud, MTX increased the extracellular space surrounding limb bud mesenchymal cells within 8–10 hours; this process continued through 16 hours and remained unabated by 24 hours. Mesenchymal cell nuclei became hyperchromatic and pyknotic during this time period. By 24 hours, a moderate amount of cellular debris was observed in the mesenchymal compartment of limb buds from approximately one-third of the embryos examined. Endothelial cell nuclei of the limb bud vasculature did not exhibit the histopathological alterations observed in the mesenchymal cells. Limb buds from embryos injected with LV at times up to 6 hours after MTX were histologically normal. When LV treatment was delayed until 16 or 20 hours after MTX, mesenchymal nuclei regained normal appearance within 2 hours of treatment; further, the abnormally large intercellular space began to decrease during the next 4 hours. Cellular debris was not a prominent feature of limb buds from LV-treated embryos examined at any time. Embryos from rabbits injected with LV at 24 hours after MTX exhibited either typical MTX-induced lesions or a sequence of reparative events similar to those described for the 16 and 20 hour LV-treated embryos. These results demonstrate that LV, administered up to 24 hours after MTX, is able to ameliorate the developmentally toxic effects of MTX seen in rabbits at term. Histological examination revealed that LV is able to reverse the early sequelae of MTX exposure in limb bud mesenchymal tissue.