Alpha-methyl CoA racemase (AMACR) is overexpressed in several human cancers, most notably colon and prostate. AMACR expression in the prostate has been investigated primarily in patients, in an older age group, treated for prostatic carcinoma and benign prostatic hypertrophy. No studies have assessed the age distribution of AMACR expression in normal men. Archival paraffin-embedded prostate tissue from 41 organ donor men (age range, 13-63 years) with no evidence of prostate neoplasia was stained with a monoclonal antibody for AMACR. Intensity was graded on a scale of 0 to 3. Semi-quantitative analysis of staining in acinar cells was used to generate a composite score (CS) [Sigma(% area x intensity)] for each case. Nondonor cases with foci of prostate cancer and high-grade prostatic intraepithelial neoplasia (PIN) were used as external positive controls for AMACR. These sections were also stained for Ki-67, to assess proliferative index. The 41 cases encompassed different age groups (13-20 years, N = 11; 20-45 years, N = 17; >45 years, N = 13). Acinar cells showed granular cytoplasmic staining. Focal positive staining was also seen in the prostatic urethra and the periurethral glands. There was wide variation in the level of expression within each age group. The level of expression seen in subjects younger than 45 years was higher (mean CS = 41.3; median CS = 22.5) than that seen in subjects older than 45 years (mean CS = 8.8; median CS = 9.0) with a P value of 0.01. Most cases in the control set of prostatic adenocarcinoma cases showed moderate to strong staining. A negative correlation was seen evaluating CS and age in subjects 20 years of age and older (r = -0.47). Ki-67 staining was variable. 1) AMACR expression can be seen in benign prostatic glandular epithelium, across all age groups. However, it is age-related, with significantly lower expression in subjects younger than 45 years. This could account for the negative staining reported in benign glands, due to biased sampling of the older population. 2) Focal positive staining is seen in the prostatic urethra and periurethral glands in 71% of the cases, with no age correlation. This is of concern because this epithelium could potentially be misinterpreted as foci of PIN. 3) The low expression of AMACR in benign glands in the older age group makes this marker useful in detecting malignancy. However, AMACR staining should be interpreted with caution and the diagnosis of PIN or prostate cancer should be rendered only with convincing histologic evidence. 4) Ki-67 staining was very variable and showed no correlation with age and AMACR expression levels. AMACR expression had no correlation with proliferative index.