Hydroxyurea (HU) inhibited DNA synthesis in the livers of partially hepatectomized rats. After an i.v. infusion of HU begun in the late G1 phase and continued for periods of up to 30 hr, all hepatocytes scheduled to embark on DNA synthesis in a characteristic intralobular sequence during this time interval accumulated at the G1-S boundary. The effective dose was 1.25 mmoles/kg/hr, preceded by a single injection of 1.69 mmoles/kg. Serum levels of HU rose slightly during continuous infusion and decreased after termination, with a half-life of about 80 min. Liver weight increased during HU infusion. The G1-S blockade was rapidly reversed in the liver after the end of HU infusion. The specific activity of DNA increased to a maximum between 3 and 5 hr. [3H]Thymidine labeling indices reached about 80%. Intralobular distribution of labeled hepatocytes was congruent to the pattern seen in partially hepatectomized rats after a continuous [3H]thymidine infusion of equal duration. The beginning of DNA synthesis in nonparenchymal cells was delayed, as compared with hepatocytes. Vincristine infusion for 12 hr after release from the HU block arrested about 40% of the hepatocytes in mitosis, indicating that a large fraction of cells progressed through the cycle after the prolonged HU block. Partially resected rat liver appeared to be rather resistant to the unfavorable consequences of "unbalanced growth" during the protracted inhibition of DNA synthesis, providing a useful model for synchronization of DNA synthesis in a differentiated resting organ triggered into active growth.