Pools of deoxyribonucleoside triphosphates in L1210 cells were assayed for drug-induced changes that might indicate the metabolic basis for retention of 1-beta-D-arabinofuranosylcytosine triphosphate by these cells after treatment with methotrexate (MTX) and 1-beta-D-arabinofuranosylcytosine (ara-C). Within 20 min after treatment with MTX, the pool of deoxythymidine triphosphate (dTTP) had decreased by about 50% and during the next 8 hr decreased slowly to 30% of its initial level. When MTX-induced decreases in the cellular contents of deoxycytidine triphosphate (dCTP), deoxyadenosine triphosphate (dATP), and deoxyguanosine triphosphate (dGTP) were normalized to percentages of the initial levels, they coincided with the second slower phase of decrease in dTTP. During the study, levels of both dTTP and dCTP remained constant in cells from mice treated with 0.9% NaCl solution, whereas levels of both dATP and dGTP decreased. MTX caused a significantly more rapid decrease in the level of dATP than did 0.9% NaCl solution but not in the level of dGTP. Over a 9-hr period, after injection of ara-C, levels of both dTTP and dCTP doubled while levels of both dATP and dGTP remained unchanged. When ara-C and MTX were administered together, levels of dTTP, dATP, and dGTP did not change significantly, and the increase in dCTP was only 25% of the increase after treatment with ara-C alone. Thus, the most striking change in deoxyribonucleoside triphosphate pools after combined administration of MTX and ara-C was an increase in dCTP concentration that reached about one-fourth the concentration achieved with ara-C alone. We suggest that MTX, by attenuating the ara-C-induced increase in dCTP, caused a change in the allosteric regulation of either deoxycytidine kinase or deoxycytidylate deaminase (or both), thereby potentiating the activity of ara-C.