INTRODUCTION: Methotrexate, a folate antagonist, is a mainstay treatment for childhood acute lymphoblastic leukemia. It is also widely used in a low dose formulation to treat patients with rheumatoid arthritis. In rats, methotrexate is known to induce micronuclei formation, leading to genetic damage, while vitamin A is known to protect against such methotrexate-induced genetic damage. Leucovorin (folinic acid) is generally administered with methotrexate to decrease methotrexate-induced toxicity. OBJECTIVES: We aimed to determine whether vitamin A and leucovorin differed in their capacity to prevent formation of methotrexate-induced micronuclei in rat bone marrow erythrocytes. The present study also aimed to evaluate the effect of combined treatment with vitamin A and leucovorin on the formation of methotrexate-induced micronuclei. METHODS: Male and female Wistar rats (n=8) were injected with 20 mg/kg methotrexate (single i.p. dose). The control group received an equal volume of distilled water. The third and fourth groups of rats received vitamin A (5000 IU daily dose for 4 successive days) and leucovorin (0.5 mg/kg i.p. dose for 4 successive days), respectively. The fifth and sixth groups of rats received a combination of vitamin A and a single dose of methotrexate and a combination of leucovorin and methotrexate, respectively. The last group of rats received a combination of leucovorin, vitamin A and single dose of methotrexate. Samples were collected at 24 hours after the last dose of the treatment into 5% bovine albumin. Smears were obtained and stained with May-Grunwald and Giemsa. One thousand polychromatic erythrocytes were counted per animal for the presence of micronuclei and the percentage of polychromatic erythrocyte was determined. RESULTS: Comparison of methotrexate-treated rats with the control group showed a significant increase in the percentage of cells with micronuclei and a significant decrease polychromatic erythrocyte percentage. Combined methotrexate and vitamin A therapy and combined methotrexate and leucovorin therapy led to significant decreases in the micronuclei percentage and an increase in polychromatic erythrocyte percentage when compared to rats treated with methotrexate alone. Leucovorin was found to be more effective than vitamin A against the formation of methotrexate-induced micronuclei. CONCLUSIONS: Both vitamin A and leucovorin provided significant protection against genetic damage induced by methotrexate.