Growing fibroblasts such as 3T3 cells are well-known to enter a quiescent state (G0) after many hours of serum deprivation. They emerge from G0 upon readdition of serum and initiate DNA synthesis about 12 h later. In this paper, we analyzed the effects of brief periods of serum deprivation on the ability of cells in G1 to initiate DNA synthesis. Exponentially growing 3T3 fibroblasts were briefly deprived of serum and their progress into S phase was monitored by autoradiography of labeled nuclei. When 10% serum was added back to cultures deprived of serum for a few hours, the progress of G1 cells into S phase was delayed for intervals far in excess of the length of the serum deprivation. Longer serum starvations resulted in longer excess delays. Several transformed 3T3 derivatives were markedly less sensitive to this serum-induced G1 regression following deprivation. When 1 microgram/ml insulin (rather than 10% serum) was added back to the starved cultures, the G1 cells entered S phase immediately. Delay in S phase entry following serum readdition was completely prevented if insulin (and, to a lesser extent, EGF) was present during the starvation, was diminished if a lower serum concentration was used for readdition, and was partially abolished if 10% serum plus insulin was restored to the cultures. The above results, then, suggest that serum deprivation sensitizes the cells to an unidentified serum component which sets the cells back in G1, unless insulin is present to maintain the flow of cells into S.