A mathematical description of polyglutamated folate kinetics for human breast carcinoma cells (MCF-7) has been formulated based upon experimental folate, methotrexate (MTX), purine, and pyrimidine pool sizes as well as reaction rate parameters obtained from intact MCF-7 cells and their enzyme isolates. The schema accounts for the interconversion of highly polyglutamated tetrahydrofolate, 5-methyl-FH4, 5-10-CH2FH4, dihydrofolate (FH2), 10-formyl-FH4 (FFH4), and 10-formyl-FH2 (FFH2), as well as formation and transport of the MTX polyglutamates. Inhibition mechanisms have been chosen to reproduce all observed non-, un-, and pure competition inhibition patterns. Steady state folate concentrations and thymidylate and purine synthesis rates in drug-free intact cells were used to determine normal folate Vmax values. The resulting average-cell folate model, examined for its ability to predict folate pool behavior following exposure to 1 microM MTX over 21 h, agreed well with the experiment, including a relative preservation of the FFH4 and CH2FH4 pools. The results depend strongly on thymidylate synthase (TS) reaction mechanism, especially the assumption that MTX di- and triglutamates inhibit TS synthesis as greatly in the intact cell as they do with purified enzyme. The effects of cell cycle dependence of TS and dihydrofolate reductase activities were also examined by introducing G- to S-phase activity ratios of these enzymes into the model. For activity ratios down to at least 5%, cell population averaged folate pools were only slightly affected, while CH2FH4 pools in S-phase cells were reduced to as little as 10% of control values. Significantly, these folate pool dynamics were indicated to arise from both direct inhibition by MTX polyglutamates as well as inhibition by elevated levels of polyglutamated FH2 and FFH2.