Although methotrexate is one of the most commonly used drugs for maintenance therapy in childhood acute lymphocytic leukemia (ALL), its oral absorption is highly variable and its intramuscular bioavailability at dosages used for ALL therapy has not been assessed in children. We therefore determined the absolute bioavailability of orally and intramuscularly administered methotrexate in 12 pediatric patients receiving 13 to 120 mg/m2 methotrexate every week as maintenance therapy for ALL. Mean bioavailability, as determined by comparing the area under the concentration-time curve after oral or intramuscular administration with that produced by the same dosage given intravenously, was 33% (range 13% to 76%) for oral (n = 11) and 76% (54% to 112%) for intramuscular (n = 7) administration (P less than 0.01). Median bioavailability (with orally administered dosages less than or equal to 40 mg/m2 (range 13 to 40 mg/m2) was 42% (19% to 76%); at dosages greater than 40 mg/m2 (43 to 76 mg/m2), bioavailability was significantly lower, 17.5% (12.7% to 22.3%, p less than 0.02). Conversely, there was no significant relationship between dosage and bioavailability with intramuscularly administered drug. The substantially higher bioavailability for intramuscularly injected methotrexate may warrant its consideration as an alternative to oral administration, especially for dosages greater than 40 mg/m2.