Expression of several growth factors is elevated in rat liver, after induction of oval cell proliferation by chemical carcinogens. However, the exact roles played by individual factors are not defined. We infused and examined the effects of epidermal growth factor (EGF) and hepatocyte growth factor (HGF) on the proliferation of ductal and periductal cells after their activation with 2-acetyl-aminofluorene (2-AAF). Furthermore, we included studies on urokinase-type plasminogen activator (uPA), because Northern blot analysis showed a strong coincidence of uPA expression with oval cell proliferation. Low doses of 2-AAF were used to activate ductal and periductal cells, whereafter growth factors were infused. Infusion of EGF, HGF, uPA, or any combination thereof for up to 7 days resulted in increased numbers of [³H]thymidine-labeled ductal and periductal cells expanding into the liver acinus. Although the growth factors all increased the number of labeled cells, they preferentially acted on different cell populations. Although exposure to 2-AAF alone or combined with infusion of HGF resulted in proliferation of almost equal numbers of ductal and Ito cells, infusion of EGF and any combination hereof resulted in 75% to 80% of labeled cells having a ductal phenotype. Also, infusion of EGF and HGF resulted in decreased numbers of cells undergoing apoptosis in response to 2-AAF. Our results demonstrate that, although 2-AAF acts as a mitogenic stimulus for ductal and periductal cells, growth factors are necessary for survival, motility, and expansion of these cells into the liver acini.