AbstractDihydrofolate reductase reduces folic acid to tetrahydrofolate as a prerequisite to one‐carbon metabolism, which is required for normal embryonic de novo DNA synthesis. The developmental toxicity of methotrexate (MTX) has been attributed to MTX's ability to inhibit the activity of dihydrofolate reductase and thereby indirectly suppress one‐carbon metabolism. The compound 1‐(p‐tosyl)‐3,4,4‐trimethylimidazolidine (TTI), which is structurally unrelated to folate, reestablishes one‐carbon metabolism by the biomimetic transfer of single carbon units. Whether the developmental toxicity of MTX is indeed caused via suppressed one‐carbon metabolism was tested in New Zealand white rabbits following concurrent maternal treatment with MTX and TTI. TTI reduced MTX developmental toxicity judged by increased mean fetal body weights, decreased percentage of malformed fetuses, and reduced incidences of major malformations. Two doses of TTI (90 mg/kg, each) at 1 hr prior to and 1 hr after MTX also reduced the developmental toxicity, but was no more effective than the single‐injection regimen. Treatment with TTI alone caused no developmental toxicity. Histologically, MTX caused enlarged intercellular spaces in limb bud mesenchyme that began at 6‐8 hr and increased in size until 16 hr. Mesenchymal nuclei appeared basophilic, with angular contours. Pretreatment with TTI delayed MTX‐induced histological changes until 20‐24 hr after MTX in 36‐50% of embryos and completely protected the remainder. The sequence of MTX‐induced changes was not altered among affected embryos, although the severity of the lesions did not appear as great. Saline‐only or TTI‐only treatments caused no alterations in limb buds. These data are consistent with the concept that impaired one‐carbon metabolism is indeed the fundamental process unde