Previous studies from this laboratory established that the rapid but partial interconversion of tetrahydrofolate cofactors to dihydrofolate after exposure of L1210 leukemia cells to antifolates cannot be due to direct feedback inhibition of thymidylate synthase by dihydrofolate or any other endogenous folylpolyglutamates when dihydrofolate reductase activity is abolished by antifolates. Rather, the data suggested this preservation of tetrahydrofolate cofactor pools is likely due to a fraction of cellular folates unavailable for oxidation to dihydrofolate. This paper explores the role of cell cycle phase in L1210 leukemia cells in logarithmic versus stationary phase growth as a factor in the rate and extent of tetrahydrofolate cofactor interconversion to dihydrofolate after exposure of cells to the dihydrofolate reductase inhibitor trimetrexate. The S phase fraction was reduced by inoculating L1210 leukemia cells at high density to achieve a stationary state. Flow cytometric analysis of DNA content indicated that log phase cultures were 53.0% S phase; this decreased to 42.1% at 24 h and 24.1% at 48 h in stationary phase cultures. 5-Bromo-2'-deoxyuridine incorporation into DNA decreased 80 and 96%, while [3H]dUrd incorporation into DNA declined 70 and 95% for stationary cultures at 24 and 48 h, respectively, as compared with the log phase rates. Log phase cells interconverted 28.0% of the total pool of radiolabeled folates to dihydrofolate with a half-time of approximately 30 s. Stationary cells at 24 h interconverted 20.4% of the total folate pool with a t1/2 of approximately 3 min, and at 48 h, net interconversion to dihydrofolate decreased further to 12.1% with a t1/2 of approximately 6 min. The decrease in the extent of tetrahydrofolate cofactor interconversion to dihydrofolate in stationary phase cells was directly proportional to the decrease in the S phase fraction determined by total DNA content. This suggests that tetrahydrofolate cofactor depletion occurs only in S phase cells. The much larger drop in [3H]dUrd and 5-bromo-2'-deoxyuridine incorporation into DNA in comparison with the decline in the S phase fraction measured by DNA content along with the reduced rate of tetrahydrofolate cofactor interconversion to dihydrofolate indicates that the rate of DNA synthesis is decreased in S phase cells in stationary cultures. Network thermodynamic simulations suggest that a reduction in the number of S phase cells and their thymidylate synthase catalytic activity would account for the observed decrease in the rate and extent of interconversion of tetrahydrofolate cofactors to dihydrofolate after trimetrexate in stationary phase cultures.(ABSTRACT TRUNCATED AT 400 WORDS)