Cyclic AMP arrests T lymphocytes in the G1 phase of the cell cycle, and prolonged exposure results in cytolysis. Both of these effects require cyclic AMP-dependent protein kinase. We recently observed that some S49 mouse T lymphoma cell lines selected for hydroxyurea resistance were not arrested in G I by cyclic AMP. Further analysis revealed that these cell lines were cyclic AMP-dependent protein kinase deficient, and conversely, other cyclic AMPdependent protein kinase deficient cell lines not selected for hydroxyurea resistance were two-to threefold more hydroxyurea resistant. However, hydroxyurea is a specific inhibitor of ribonucleotide reductase and does not inhibit this kinase. We subsequently showed that cyclic AMP-dependent protein kinase will phosphorylate the M 2 but not the M1 subunit of ribonucleotide reductase in vitro, and this phosphorylation will diminish CDP reductase activity. In vivo phosphorylation of M 2 occurred under conditions similar to those that generate cell cycle arrest. We conclude that the M 2 subunit of ribonucleotide reductase can be a target of cyclic AMP-dependent protein kinase. The phosphorylated enzyme has diminished activity, and this may play a role in cyclic AMP-induced lymphocyte cell cycle arrest.