The ability of L1210 mouse leukemia cells and of a mutant methotrexate-resistant cell line (L1210/MTX) to synthesize methotrexate polyglutamates was studied. Host DBA/2 mice were treated with methotrexate, after which leukemic cells were harvested from ascitic fluid and levels of methotrexate and metabolites in them were determined by Sephadex G-15 gel chromatography. The level of methotrexate in L1210/MTX cells was 12.5 times greater than that in L1210 cells, reflecting the increased level of dihydrofolate reductase that characterizes this mutant cell line. Synthesis of methotrexate polyglutamates in each cell line required a dose of methotrexate (2.4 mg/kg) 10 times greater than the dose that yielded extensive methotrexate polyglutamate synthesis in rat liver and kidney in previous studies. Total methotrexate polyglutamates synthesized in 4 hr with this dose were the same in each cell line, demonstrating that this metabolism was not affected by differences in the level of dihydrofolate reductase. Methotrexate polyglutamates comprised 47+/-20% of the total methotrexate in L1210 cells. Methotrexate diglutamate was the predominant form. Levels of methotrexate monoglutamate and diglutamate were similar in L1210/MTX cells, whereas methotrexate monoglutamate was the predominant metabolite in host liver, kidney, and small intestine. These differences may reflect differences in substrate preference of pteroylpolyglutamate synthetase in these different tissues. Twenty-four hr after methotrexate administration, total methotrexate in L1210 cells was one-third of that at 4 hr; but the proportion of metabolites was the same, presumably reflecting cell death and division rather than loss of a freely exchangeable portion of intracellular methotrexate present at the earlier time. The affinity of methotrexate polyglutamates for dihydrofolate reductase was found to be similar to that of methotrexate, providing evidence that these metabolites may be as potent antagonists of folate metabolism as is methotrexate itself. Recent studies indicate that inhibition of folate metabolism in cells requires their exposure to high levels of methotrexate in order to achieve intracellular levels of methotrexate greater than needed to bind to dihydrofolate reductase. Such conditions conform to those required for synthesis of methotrexate polyglutamates. Thus, these metabolites may play a specific role in inhibiting folate metabolism, distinct from the antifolate potential that they appear to share with methotrexate.