Experiments were carried out in L1210 cells to examine the importance of 'substrate cycles' in regulating the intracellular levels of deoxyribonucleoside 5'-triphosphate. L1210 cells were incubated with [14C]cytidine or [14C]adenosine in the presence and absence of hydroxyurea or cytosine arabinoside (araC). These incubations were carried out for either 30 or 120 min. Inhibition of ribonucleotide reductase by hydroxyurea resulted in the blockage of the flux of ribonucleotides to deoxyribonucleotides (greater than 90%) as expected. When DNA synthesis was inhibited with araC, there was a marked decrease in the incorporation of [14C]cytidine or [14C]adenosine into DNA as deoxyribonucleotides. However, there was not a corresponding increase in the deoxyribonucleotide levels in the acid-soluble fraction or deoxyribonucleosides in the culture medium. AraC treatment decreased the total formation of deoxyribonucleotides. These data indicate that L1210 cells do not regulate the intracellular pools of dNTPs via 'substrate cycles' which involve activation of phosphatases when DNA synthesis is blocked or activation of kinases when ribonucleotide reductase is inhibited.