Purpose We evaluated the role of tissue inhibitor of metalloproteinase-1 (TIMP-1) in the invasion and metastasis of human urothelial cancer. Materials and Methods The expression level of TIMP-1 messenger (m)RNA was determined in 33 urothelial carcinomas, including upper urothelial cancer in 15 cases and bladder cancer in 18, by Northern blot analysis. Localization of TIMP-1 mRNA was analyzed by in situ hybridization. These data were compared with clinicopathological features. We also determined the growth activity of tumor cells using immunohistochemical staining for Ki-67 and compared it with the expression levels of TIMP-1 mRNA. These data were also compared with clinicopathological features. Results The expression level of TIMP-1 mRNA significantly correlated with pathological stage and histological differentiation. In situ hybridization showed that TIMP-1 mRNA was expressed in the cytoplasm of urothelial cancer cells. The Ki-67 labeling index significantly correlated with the expression level of TIMP-1 mRNA in urothelial cancer. Patients with high expression of TIMP-1 mRNA had a poorer prognosis than those with low expression. Conclusions TIMP-1 mRNA is expressed in the cytoplasm of cancer cells and high expression indicates more malignant potential in cases of urothelial cancer.