Recent studies have suggested that matriptase, a transmembrane serine protease and its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) are important in the progression of many cancers. Limited quantitative data are available on these proteins in prostate cancer. To validate the roles of matriptase and HAI-1 in prostate cancer and its progression, a prostate cancer tissue microarray was constructed. The tissue microarray includes 41 localized prostate cancers (Pca_local), 18 aggressive prostate cancers, 18 metastatic prostate cancers, 24 benign prostate hyperplasias, 18 high-grade intraepithelial neoplasias (HGPIN), and 41 benign prostate tissues. The cellular expression levels of matriptase and HAI-1 were quantified using automated quantitative analysis. We found that matriptase expression levels were significantly higher in Pca_local (P<0.0001) and HGPIN (P<0.05) compared with benign prostate tissue. Matriptase levels were significantly decreased in metastatic cancer when compared with all other tissue types (P<0.05). Compared with benign prostate tissue, HAI-1 expression levels were significantly higher in all proliferative prostate diseases (benign prostate hyperplasia, HGPIN, localized and aggressive cancers, and metastases) (P<0.001); yet, no significant differences were found in HAI-1 expression levels among the diseased tissue types. These results suggest that an increase of matriptase may be useful as a marker for detection of Pca_local, whereas a decrease of matriptase expression may signal prostate cancer progression. HAI-1 seems to be a marker of prostate epithelial cell proliferation.