The purpose of this study was to retrospectively characterize benign and malignant prostate peripheral zone tissue by using endorectal MRI and 3D 1H MRS. Fifty-two men with untreated biopsy-proven prostate cancer underwent combined endorectal MRI and MRSI. Whole-mount step-section histopathologic analysis constituted the reference standard. Biopsy correctly detected 74 locations; MRI correctly detected 72 locations; MRS correctly detected 72 locations; MRI + MRS correctly detected 78 locations. Cohen’s test showed that biopsy had a lower degree of agreement with histology than MRI + MRS combined. The ratio of [(Cho + Cr)]/Cit correlates with the pathologic Gleason score. The addition of 3D1H MRSI to MRI can improve diagnosis of prostate cancer contributing indirectly to improve local staging. In addition, the correlation between metabolic 3D1H MRSI data with pathological Gleason grade may offer a non-invasive means to better predict prostate cancer aggressiveness.