Prostate cancer (PCA) is the most common type of cancer found in men of western countries and is the leading cancer death next to lung cancer and colorectal cancer. Prostate-specific antigen (PSA) test is an established diagnostic tool for PCA detection, but confirmation of diagnosis by histopathological evaluation of prostate needle biopsies is performed. To define protein expression pattern of prostate biopsies, in the present study we investigated biopsy samples from benign prostate hyperplasia (BPH, n=11) and prostate cancer (PCA, n=12) patients by two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify potential biomarkers which might distinguish the two clinical situations. 2-DE results revealed 88 protein spots expressed differentially among hyperplasia and cancer groups with statistical significance. Interesting spots were analyzed by MALDI-TOF-MS-MS and 79 different proteins were identified. The important proteins identified included prostatic acid phosphatase precursor, a significant overexpressed protein in PCA, prohibitin, NDRG1 tumor suppressor proteins, heat shock proteins, cytoskeletal proteins, enzymes like DDAH1 and ALDH2. Prohibitin was investigated in detail at mRNA level and protein level using immunohistochemistry on prostatectomized specimens. We found that the level of mRNA for prohibitin correlates with the increased amount of protein indicating involvement of changes at transcriptional level. Furthermore, immunohistochemistry revealed no staining in BPH (n=13), moderate staining in prostate intra-epithelial neoplasia (PIN, n=5) but strong staining in PCA (n=18). Our results demonstrate that protein profiling and mRNA studies can be performed on the same prostate biopsy. Moreover, our study revealed a significant up-regulation of prohibitin in prostate cancer compared to BPH which may be a potential marker to distinguish PCA and BPH. Some of the interesting proteins identified in this approach may serve to develop new targets for PCA diagnosis and treatment.