Hydroxyurea inactivates ribonucleotide reductase from mammalian cells and thereby depletes them of the deoxynucleoside triphosphates required for DNA replication. In cultures of exponentially growing 3T6 cells, with 60-70% of the cells in S-phase, 3 mM hydroxyurea rapidly stopped ribonucleotide reduction and DNA synthesis (incorporation of labeled thymidine). The pool of deoxyadenosine triphosphate (dATP) decreased in size primarily, but also the pools of the triphosphates of deoxyguanosine and deoxycytidine (dCTP) were depleted. Paradoxically, the pool of thymidine triphosphate increased. After addition of hydroxyurea this pool was fed by a net influx and phosphorylation of deoxyuridine from the medium and by deamination of intracellular dCTP. An influx of deoxycytidine from the medium contributed to the maintenance of intracellular dCTP. 10 min after addition of hydroxyurea, DNA synthesis appeared to be completely blocked even though the dATP pool was only moderately decreased. As possible explanations for this discrepancy, we discuss compartmentation of pools and/or vulnerability of newly formed DNA strands to nuclease action and pyrophosphorolysis.