System A, the sodium-dependent neutral amino acid transport activity, has a 3-fold increase in its initial uptake velocity into hepatocytes following partial hepatectomy (PH) in the rat. The purpose of this study was to examine the effect of inhibition of System A–mediated amino acid transport on hepatocyte proliferation and liver regeneration. We describe thein vivocompetitive inhibition of System A activity following PH by the nonmetabolizable, System A–specific substrate, α-(methylamino)isobutyric acid (MeAIB). Administration of MeAIB 60 minutes before PH decreased the incorporation of [3H]thymidine into DNA by 45% ± 5% and 76% ± 17% at 24 and 36 hours, respectively. The readministration of MeAIB every 12 hours further decreased DNA synthesis by 92% ± 18% and 82% ± 11% at 24 and 36 hours. The recovery of liver mass of rats receiving MeAIB was decreased by 46.4% ± 5.1% at 24 hours after PH.In vitro, 5 mmol/L MeAIB inhibited proliferation of primary hepatocytes by 56% ± 4% and 61% ± 12% 48 hours after incubation with 10% fetal calf serum or epidermal growth factor (5 ng/mL), respectively. Thus, MeAIB inhibition of System A transport activity decreased bothin vivoand in vitroinducement of hepatocyte proliferation. Treatment with MeAIB did not significantly change the incorporation of [3H]leucine into total liver protein, but changes in serum amino acids and hepatocyte cell volume were observed, suggesting System A transport activity during hepatocyte proliferation functions primarily to provide amino acids to fuel liver-specific biochemical pathways and to increase cell volume.