Determinants of the timing of DNA synthesis in rat liver were studied, using alpha-hexachlorocyclohexane as a tool for stimulation of cell proliferation. One determinant is the time of alpha-hexachlorocyclohexane administration. The increase in DNA synthesis starts after a lag phase (prereplicative phase) of minimally 20 hr. Use of animals adapted to a controlled feeding and lighting schedule revealed a second determinant provided by food consumption. Initiation of DNA synthesis is suppressed by fasting or protein deprivation and occurs 5 to 8 hr after readministration of a protein-containing diet. The light-dark rhythm has no direct influence on the timing of DNA synthesis. Stimulation of hepatic DNA synthesis, therefore appears to require two different sequential signals. The first is provided by alpha-hexachlorocyclohexane, and the second is provided by protein intake. In the absence of the second signal, committed cells are arrested at a critical point of the prereplicative phase and accumulate. Protein intake permits release from the block, and the accumulated cells enter the S period almost synchronously after completion of the remaining 5 to 8 hr of the prereplicative phase. These observations provide a means of synchronizing, in the living animal, a proliferating population of hepatocytes. In addition, they offer an explanation for the diurnal rhythmicity in the rate of hepatic cell proliferation.