In the present study we have examined the immunohistochemical expression of matrix metalloproteinases (MMPs) in bile duct cancer tissue and the relationship between MMP expression and various clinicopathologic factors. We performed immunohistochemical studies of matrix metalloproteinase-2 (MMP2), and matrix metalloproteinase-3 (MMP3). MMP2 and MMP3 were expressed in 48.9% (23/47) and 44.7% (21/47) of the subjects, respectively. Subjects positive for MMP2 showed significantly higher association rates of MMP3 expression than those who were not (p<0.05). MMP2 expression was also significantly higher in the group with histologically well-differentiated lesions than in the group with poorly differentiated lesions (p<0.05). Expression of MM2 and MM3 was significantly higher in subjects with neural invasion (p<0.05). The MMP2-negative group had a significantly better prognosis than the MMP2-positive group (p<0.05). MMP2 expression tended to increase as the degree of differentiation decreased, and this indicated a possibility that MMP2 and MMP3 may be involved in the development of perineural invasion in bile duct cancer. Perineural invasion is an important prognostic factor in bile duct cancer, and the fact that various types of cytokines are involved in the activation of MMPs suggested that bile duct cancer complicated by obstructive cholangitis might be associated with a poorer prognosis.