We investigated the cell cycle regulation of deoxyribonucleoside triphosphate (dNTP) metabolism in hydroxyurea-resistant (HYUR) murine S49 T-lymphoma cell lines. Cell lines 10-to 40-fold more hydroxyurea-resistant were selected in a stepwise manner. These HYUR cells exhibited increased CDP reductase activity (5-to 8-fold) and increased dNTP pools (up to 5-fold) that appeared to result from increased activity of the M2 subunit (binding site of hydroxyurea) of ribonucleotide reductase. These characteristics remained stable when the cells were grown in the absence of hydroxyurea for up to 2 years. In both wild type and hydroxyurea-resistant cell populations synchronized by elutriation, dCTP and dlTP pools increased in S phase, whereas dATP and dGTP pools generally remained the same or decreased, suggesting that allosteric effector mechanisms were operating to regulate pool sizes. Additionally, CDP reductase activity measured in permeabilized cells increased in S phase in both wild type and hydroxyurea-resistant cells, suggesting a nonallosteric mechanism of increased ribonucleotide reductase activity during periods of active DNA synthesis. While wild type S49 cells could be arrested in the GI phase of the cell cycle by dibutyryl cyclic AMP, hydroxyurea-resistant cell lines could not be arrested in the G I phase by exogenous cyclic AMP or agents that elevate t h e concentration of endogenous cyclic AMP. These data suggest that cyclic AMP-generated GI arrest in S49 cells might be mediated by the M2 subunit of ribonucleotide reductase.