Hepatocyte growth factor (HGF), a ligand of c-Met receptor, stimulates activation of cellular kinases via phosphatidylinositol 3-kinase (PI3-kinase). CCAAT/enhancer binding protein (C/EBP) controls cell cycle progression. The present study was designed to determine whether HGF activates C/EBP in association with the S-phase entrance for cell replication and whether PI3-kinase contributes to the activation of C/EBP. Treatment of H4IIE cells, a hepatocyte-derived cell line, with HGF increased protein binding to the C/EBP binding site at an early time. Immunodepletion, subcellular fractionation, and confocal microscopic analyses showed that the HGF-induced C/EBP DNA binding activity depended on nuclear translocation of C/EBP beta. Whereas stable transfection of the p110 catalytic subunit of PI3-kinase enhanced HGF-mediated nuclear translocation of C/EBP beta and DNA binding, stable transfection of p85 subunit or chemical inhibition of PI3-kinase completely blocked C/EBP activation. HGF increased luciferase reporter activity in cells transfected with a mammalian cell expression vector containing -1.65 kilobase rGSTA2 promoter comprising C/EBP response element (pGL-1651). Whereas transfection with pCMV500, a control vector, allowed pGL-1651 to respond to HGF, expression of dominant negative mutant C/EBP completely inhibited the ability of HGF to stimulate the reporter gene expression. Flow cytometric analysis showed that HGF caused an increase in the area of S phase with a reciprocal decrease in that of G(1) phase, suggesting that HGF promoted cell cycle progression to S phase. In conclusion, HGF induces nuclear translocation of C/EBP beta via the PI3-kinase pathway and stimulates C/EBP DNA binding and gene transcription and that the PI3-kinase-mediated C/EBP activation by HGF may contribute to cell replication.