Hypermethylation of the 5' promoter region of the glutathione S-transferase pi gene (GSTP1) occurs at a very high frequency in prostate adenocarcinoma. We compared the results of blinded histologic review of sextant biopsy samples from 72 excised prostates with those obtained using a quantitative methylation-specific polymerase chain reaction assay (QMSP) for GSTP1. Formal surgical pathologic review of the resected prostates was used to determine the number of patients with (n = 61) and without (n = 11) prostate cancer. Histology alone detected prostate carcinoma with 64% sensitivity (95% confidence interval [CI] = 51% to 76%) and 100% specificity (95% CI = 72% to 100%), whereas the combination of histology and GSTP1 QMSP at an assay threshold greater than 10 detected prostate carcinoma with 75% sensitivity (95% CI = 63% to 86%) and 100% specificity (95% CI = 72% to 100%), an 11% improvement (95% CI = 5% to 22%) in sensitivity over histology alone. The combination of histology and GSTP1 QMSP at an assay threshold greater than 5 detected prostate adenocarcinoma with 79% sensitivity (95% CI = 68% to 89%), a 15% improvement (95% CI = 7% to 26%) over histology alone. Thus, GSTP1 QMSP improved the sensitivity of histologic review of random needle biopsies for prostate cancer diagnosis. Further studies should determine whether detection of GSTP1 hypermethylation in a biopsy sample with normal histology indicates the need for an early repeat biopsy at the same site.