2'-Deoxy-2'-azidocytidine inhibits DNA synthesis of cells in tissue culture. In vitro, the diphosphate of azidocytidine was earlier shown to inhibit mammalian ribonucleotide reductase, while the triphosphate did not inhibit DNA replication in isolated cell nuclei. In intact cells, however, deoxyribonucleoside triphosphate pools were not decreased by azidocytidine in a manner expected from an inhibitor of ribonucleotide reductase. In order to differentiate between inhibition of precursor synthesis and DNA replication we studied in the present investigation the effects of azidocytidine on pool sizes of deoxynucleotides as well as on the incorporation of labeled cytidine or deoxycytidine into deoxycytidine triphosphate pools and DNA of cultured growing mouse fibroblasts. Azidocytidine was transformed to an active metabolite by phosphorylation with deoxycytidine kinase. Cell lines deficient in this enzyme showed an increased resistance against the analogue. Azidocytidine had a dual effect on the incorporation of labeled nucleosides. At low concentrations, the incorporation of cytidine into DNA was severely inhibited without a decrease of the specific activity of the dCTP pool or the size of the other three dNTP pools, suggesting a direct effect on DNA replication. At high concentrations of azidocytidine, incorporation into dCTP was also affected, demonstrating inhibition of CDP reduction. Our data also show that reduction of CDP is not necessarily tightly coupled to DNA replication since a rapid turnover of the dCTP pool occurred at the low concentrations of azidocytidine which severely inhibited DNA synthesis.