Hepatic stellate cells (HSC) differ in their phenotype depending on the initiation and progression of their activation. Our hypothesis was that different mechanisms govern type I collagen synthesis depending on stage of HSC activation. We investigated the role of α5β1-integrin as a regulator of type I collagen gene COL1A1 expression in primary and passaged HSC cultures using transgenic mouse containing type I collagen gene COL1A1 promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The α5β1protein levels increased during the activation and were highest in day 6 primary cultures but decreased in passaged HSC. CAT activity, reflecting COL1A1 expression, was upregulated by α5β1-integrin. Inhibition of α5β1-integrin by echistatin and blocking antibody resulted in reduced transgene activity only in early primary cultures (compared with the control, 53.3 ± 12% echistatin and 58.8 ± 7% blocking antibody, respectively, P < 0.05). Treatment of passaged HSC with either echistatin or blocking antibody had no effect. Fibronectin, an α5β1-integrin ligand, increased transgene activity in primary (210 ± 33%, P < 0.05) but not in passaged HSC cultures (119 ± 8%). This α5β1-integrin effect appears to be at least in part mediated by CCAAT enhancer binding protein-β (C/EBPβ), because fibronectin increased and α5-gene silencing by small interfering RNA decreased C/EBPβ levels. In addition, C/EBPβ knockout mice showed reduced type I collagen synthesis compared with wild-type littermates. Therefore α5β1-integrin is an important regulator of type I collagen production in early primary HSC cultures but appears to have no direct role once the HSC are fully activated.