Methotrexate (MTX) and 6-mercaptopurine (6MP) are common drugs in the oral maintenance therapy of acute lymphoblastic leukemia (ALL). On the basis of their biochemical effects on cell metabolism, a sequence-dependent synergism might be anticipated. In order to investigate this hypothesis, MOLT-4 human malignant T-lymphoblasts were incubated with various concentrations of MTX. The time at which maximal increase of intracellular 5-phosphoribosyl-1-pyrophosphate (PRPP) levels was found correlated with the concentrations of MTX used. Determination of aminoimidazolecarboxamide ribonucleoside monophosphate (AICAR) levels and labeled glycine incorporation into purine metabolites revealed an incomplete inhibition of purine de novo synthesis after incubation with 0.02 microM MTX, and a complete inhibition with 0.2 microM MTX. After prolonged periods of incubation, glutamine exhaustion of the medium caused inhibition of purine de novo synthesis in MTX-untreated cells, with a concomitant increase of PRPP levels. Addition of glutamine to the medium prevented this phenomenon. The increased availability of PRPP after pretreatment with MTX can be used for enhanced intracellular incorporation of hypoxanthine and 6MP in their respective nucleotides. The time- and dose-dependent effects of MTX on PRPP levels correlated with the enhanced incorporation of hypoxanthine and 6MP. The data presented in this study demonstrate that a synergistic action of the combination of MTX and 6MP can be anticipated in malignant lymphoblasts with an active purine de novo synthesis depending on the concentration of MTX and on the time and sequence of administration of both drugs.