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 alpha5beta1(51) 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 CAT reporter gene. 51 protein 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 51 integrin. 51 integrin inhibition by echistatin and blocking antibodies resulted in reduced transgene activity only in early primary cultures (compared to the control 53.3±12% echistatin and 58.8±7% blocking antibodies respectively, p<0.05). Treatment of passaged HSC with either echistatin or blocking antibodies had no effect. Fibronectin, an 51 integrin ligand, increased transgene activity in primary (210±33%, p<0.05) but not in passaged HSC cultures (119±8%, p>0.05). This 51 integrin effect appears to be at least in part mediated by CCAAT enhancer binding protein beta (C/EBP ), as fibronectin increased and 5 gene silencing by siRNA decreased C/EBP levels. In addition, C/EBP knockout mice showed reduced type I collagen synthesis when compared to wild type littermates. Therefore, 51 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.