Human gallbladder cancer is highly malignant and its prognosis is usually poor depending on the extent of surrounding tissue invasion. We examined in vitro the invasive activity of four gallbladder cancer cell lines (GB-d1, GB-h3, GB-d2 and FU-GBC-1) in the absence or presence of hepatocyte growth factor (HGF). In type 1 collagen gel culture, HGF stimulated cell proliferation and induced an invasive phenotype of arborizing structures in GB-d1, GB-h3 and GB-d2. In a Matrigel invasion assay, invasion was also induced in three of these cell lines by HGF but not in FU-GBC-1. Cellular motility was, however, stimulated by HGF in all of the four cell lines to various extents. Zymography for proteolytic enzymes demonstrated high levels of type IV collagenase and urokinase-type plasminogen activator (u-PA) activity in GB-d1, GB-h3 and GB-d2 even in the absence of HGF. In the presence of HGF, the 72 kDa type IV collagenase (MMP-2) activity of GB-h3 and u-PA activities of GB-d1, GB-h3 and GB-d2 were enhanced. In contrast, the MMPs and PAs activities of FU-GBC-1 were faint irrespective of the addition of HGF. A Western blot analysis demonstrated higher levels of 190 kDa c-MET product (HGF receptor) of GB-d1, GB-h3 and GB-d2 than that of FU-GBC-1. The invasion in the Matrigel assay stimulated by HGF was inhibited by protease inhibitors, aprotinin and FOY-305, as well as by anti-HGF antibody. These results thus suggest that, in addition to the importance of the proteolytic activity, the cellular motility induced via the HGF/HGF-receptor system is essential for the invasive progression of gallbladder carcinoma cells.