Ribonucleotide reductase is responsible for the production of the deoxyribonucleotides required for DNA synthesis. The enzyme is composed of two dissociable subunits, proteins M1 and M2, which are inactive alone, but are fully active when combined. From mouse S49 T lymphoma cells we have isolated and separated the two subunits and used each for determining the activity of the complementary subunit in extracts from cells of different phases in the cell cycle. Treatment of S49 cells with cAMP analogs (e.g. Bt2cAMP) results in the protein kinase-dependent arrest of the cells in the G1 phase of the cell cycle. Ribonucleotide reductase (holoenzyme) activity fell in S49 cells treated for more than 16 h with Bt2cAMP but was unchanged during short term treatments. The activity of protein M2 was decreased in parallel to the overall activity of ribonucleotide reductase, while protein M1 activity changed less. Removal of bt2cAMP after 24 h exposure resulted in increased holoenzyme and protein M2 activities. Centrifugal elutriation of exponentially growing S49 cells separated cells into a 90% pure G1 cell population a mixture of G1 and early S phase cells and a 95% pure S phase/G2 cell population. The specific catalytic activity of protein M1 was the same in all these fractions while that of protein M2 was decreased 60% in the G1 cell population. These results demonstrate that the ribonucleotide reduction necessary for DNA synthesis is regulated in a cell cycle-dependent fashion by the activity of the protein M2 subunit of ribonucleotide reductase.