The basis for the proliferation-dependent cytotoxicity of methotrexate has been investigated in mice bearing the L5178Y ascites leukemia. Methotrexate at 60 mg/kg i.p. reduced the viability of logarithmically growing ascites cells (55% active S phase cells) to 28% of control, whereas the viability of the slowly growing cells (18% active S phase) was decreased to only 59% of control. Log phase tumor cells accumulated 8-fold higher levels of methotrexate polyglutamates compared to cells that had approached the stationary phase. However, no differences between log phase and slowly growing tumor cells were observed in the cellular levels of unmetabolized methotrexate. Intestinal mucosa and bone marrow from non-tumor-bearing mice resembled slowly growing tumor cells and had markedly lower levels of methotrexate polyglutamates than logarithmically growing cells. The greater accumulation of methotrexate polyglutamates in the logarithmically growing tumor cells was consistent with an increased synthesis of methotrexate polyglutamates in these cells. The enhanced methotrexate polyglutamylation in log phase versus slowly growing cells was not related to changes in the rates of either cellular methotrexate transport, transmembrane efflux of methotrexate, or hydrolysis of methotrexate polyglutamates. Thymidylate synthase activity measured in situ and in extracts from log phase cells was 4- and 2-fold higher, respectively, than in the more slowly growing cells. Methotrexate produced a 2.4-fold greater depletion of poly-gamma-glutamyl derivatives of 5,10-methylenetetrahydropteroylglutamate in log phase cells compared to slowly growing cells, and this was a function of both the increased methotrexate polyglutamate accumulation and thymidylate synthase activity in the rapidly proliferating cells. These results provide further evidence that the selectivity of methotrexate for tumors with a high growth fraction is a consequence of the rapid rates of both cellular methotrexate polyglutamate synthesis and oxidation of 5,10-methylenetetrahydropteroyl polyglutamates by thymidylate synthase.