Previous studies in which investigators have induced the rate of polyamine uptake in vitro have used either inhibitors of polyamine biosynthesis or growth factors that induce cell proliferation. Recently, however, we have described the induction of putrescine uptake in cultured adult mouse hepatocytes and have shown that uptake is independent of both intracellular polyamine levels and proliferation. Although proliferation was not apparent in those studies, data suggested that, after isolation, the cells entered G1 of the cell cycle. In this study, we have examined whether the induction of putrescine uptake is a function of entry into the cell cycle and whether uptake activity is essential for optimal progression into the S phase. Using ribonuclease reductase subunit M1 as a marker of entry into the cell cycle, we have shown that hepatocytes enter G1 during the first 4 hr of culture. Both putrescine uptake and ornithine decarboxylase activity increased as the cells entered G1. Treatment of the cells with retinoic acid (10 to 33 μmol/L) prevented them from entering G1 and also inhibited the induction of the putrescine transporter by up to 90%. In contrast, initiation of G1 to S phase transition markedly down-regulated the activity of the transporter. Thus induction of the putrescine transporter in isolated hepatocytes appears to be a G1-specific event. Culturing the hepatocytes in the presence of 1,1′-bis[3-(1′-methyl-[4,4′-bipyridinium]-1-yl)-propyl]-4,4′-bipyridinium, a potent competitive inhibitor of putrescine uptake, resulted in a 47% decrease in intracellular putrescine. Measurement of the distribution of tracer 3H polyamines showed a loss of intracellular polyamines and an accumulation of extracellular polyamines when cells were treated with 1,1′-bis[3-(1′-methyl-[4,4′-bipyridinium]-1-yl)-propyl]-4,4′-bipyridinium, indicating that the re-uptake of effluxed polyamines contributes to intracellular polyamine homeostasis in cultured hepatocytes. DNA synthesis was significantly inhibited in 1,1′-bis[3-(1′-methyl-[4,4′-bipyridinium]-1-yl)-propyl]-4,4′-bipyridinium–treated cells, and this effect was completely reversed by the addition of 200 μmol/L extracellular putrescine. We concluded that putrescine uptake is important for maintaining high intracellular putrescine levels required for optimal G1 to S phase transition in isolated mouse hepatocytes. (HEPATOLOGY 1991;14:1243–1250.)