The chemotherapeutic value of methotrexate resides in its ability to perturb folate-dependent one-carbon metabolism and subsequently inhibit DNA synthesis. To assess the functional effect of methotrexate on hepatic one-carbon metabolism, we have developed and applied tracer kinetic techniques in vivo to quantify the carbon flux through the folate-dependent one-carbon pool in rats. Following a 7-day treatment period with methotrexate (0.2 mg/kg body weight), the tracers L-[ ring -2 ^-l4 C] histidine and L-[ methyl - ³ H] methionine were simultaneously infused into control and methotrexate-treated rats. Methotrexate treatment decreased hepatic tetrahydrofolate, methyltetrahydrofol-ate, and formyltetrahydrofolate concentrations by 63, 83, and 58%, respectively. Furthermore, the enzymatic activity of 10-formyltetrahydrofolate dehydrogenase, the terminal enzyme in the catabolism of the ring -2-carbon of histidine to CO ₂ , was diminished by 32% in methotrexate-treated animals. These changes in enzyme activity and folate coenzyme concentrations did not result in a significant decrease in the oxidative flow of carbon from histidine to CO2 in methotrexate-treated rats compared to control animals (2.40 and 3.22 (imol/h/kg3/4, respectively). Oxidative carbon flow was reflective of tetrahydrofolate and formyltetrahydrofolate pools when expressed as a percent of total folate: neither coenzyme pool was diminished as a result of methotrexate treatment In contrast, the reductive carbon flux through the one-carbon pool from histidine to methionine was significantly decreased 59% in methotrexate-treated (7.63 μmol/h/kg ^¾ ) versus control rats (18.73 nmolAh/kg3/4)). Likewise, methyltetrahydrofolate, as a percent of total folate, was reduced 51% in methotrexate-treated rats. Consequently, total measured carbon flow (oxidative + reductive) was 54% lower in rats subjected to subchronic methotrexate treatment These tracer kinetic experiments quantitatively demonstrate the extent to which methotrexate alters the actual carbon flow through the hepatic folate-dependent one-carbon pool, primarily directed at diminishing the reductive carbon flow towards methyltetrahydrofolate and methionine synthesis.