Stellate cells play an important role in the production and turnover of the normal extracellular matrix of the liver and are key effector cells in the hepatic fibrogenesis that occurs in response to liver injury. In the present study, we used a rat model of long term dietary iron supplementation to identify stellate cell genes that are expressed during chronic hepatic iron overload. Using a subtraction cloning strategy, we identified a rat isoform of the complement C4 protein gene whose expression was strongly induced in stellate cells after iron overload. Highly purified, cultured stellate cells synthesized the C4 precursor protein and released its subunits into the culture medium. The C4 protein secreted in vitro was biologically active in a C4-specific hemolytic assay. C4 mRNA expression was minimal in freshly isolated stellate cells and increased between days 3 and 7 of primary culture, coincident with the expression of smooth muscle alpha-actin (alpha-SMA), a marker of cellular activation. C4 expression was absent in strongly alpha-SMA-positive, passaged cells, but was induced by IFN-gamma, which simultaneously inhibited alpha-SMA expression. Our studies establish hepatic stellate cells as a previously unrecognized source of C4 and raise the possibility that complement protein expression by the cells plays a role in the hepatic injury response and in fibrogenesis. Our in vitro data point to the presence of two distinct stimulatory pathways for C4 expression in stellate cells that differ with regard to their sensitivity to IFN-gamma and their relationship to cellular activation.