Amongst the mechanisms known to mediate resistance to methotrexate (MTX), a major component in the treatment of childhood leukemia, reduced drug accumulation appears to have direct clinical relevance. However, due to the poor viability of patient-derived acute lymphoblastic leukemia cells in vitro, determination of this parameter in clinical samples is associated with a number of difficulties. We have therefore developed an assay for reduced MTX accumulation, which controls for the metabolic state of the cell population under study by utilizing accumulation of the nucleoside thymidine as an independent indicator of this parameter. To establish this assay, we have utilized pediatric leukemic cell populations maintained as xenografts in nude mice, which, despite displaying sensitivity to MTX, demonstrated reduced accumulation of MTX when assayed using standard methodology. When accumulation of MTX by such cell populations was expressed, however, relative to their accumulation of thymidine, MTX accumulation was shown to be equal to that of drug-sensitive CCRF-CEM cells maintained in long-term culture. In contrast, significantly less MTX was accumulated, in this assay, by xenografted cell populations with demonstrated resistance to MTX. Identical results were obtained using either fresh or cryopreserved cells. The data thus indicate that by controlling for variable metabolic status of leukemic cells, it is possible to accurately assess MTX accumulation in leukemic samples displaying limited viability in culture.