Objectives: This study was designed to determine whether citrate levels detected by localized 1H spectroscopy could reliably discriminate regions of prostate adenocarcinoma from surrounding regions of normal peripheral zone and benign prostatic hyperplasia (BPH). Methods: In 28 patients and 5 volunteers stimulated echo proton spectroscopy was used in conjunction with endorectal surface coils to obtain water-suppressed 1H spectra from regions of normal prostate peripheral zone, BPH, and prostate cancer. 1H spectra from prostate cancer patients were correlated with pathologic areas identified on T2-weighted endorectal coil magnetic resonance (MR) images and histologic study of the step-sectioned gland after surgery. Results: The major finding of in vivo studies was consistently lower citrate levels in prostate cancer compared with BPH and normal prostate peripheral zone. This was reflected by significantly (P < 0.05) lower mean citrate/(creatine plus choline) peak area ratio observed for regions of cancer (0.67 ± 0.17) compared with BPH (1.21 ± 0.29) and normal peripheral zone (1.46 ± 0.28). Moreover, there was no overlap of individual cancer and normal peripheral zone citrate ratios and no significant difference between citrate ratios in regions of normal peripheral zone in young volunteers (1.28 ± 0.14) and age-matched patients (1.46 ± 0.28). The observed alterations in vivo citrate levels were supported by citrate concentration data obtained from extracts of histologically proven samples of normal, benign, and malignant prostatic tissues removed at surgery. In vitro citrate levels in the normal peripheral zone (30.9 ± 8.5 μmol/g wet weight) and BPH (46.3 ± 5.4 μmol/g wet weight) were significantly higher than those for prostate cancer (3.74 ± 0.54 μmol/g wet weight). Conclusions: These studies further demonstrate the potential of citrate as an in vivo marker for discriminating prostate cancer from surrounding regions of normal peripheral zone and BPH.