The lignan enterolactone, produced by the intestinal microflora from dietary precursors, has been hypothesized to protect against hormone-dependent cancers and cardiovascular diseases. We conducted a nested case-control study to examine the relationship between serum enterolactone concentration and prostate cancer. Enterolactone concentrations were measured by time-resolved fluoroimmunoassay in serum collected at baseline in the alpha-Tocopherol, beta-Carotene Cancer Prevention Study from 214 men with prostate cancer diagnosed during a 6-year follow-up and from 214 controls matched by age, date of baseline blood collection, intervention group, and local study area. Mean serum enterolactone concentration (in nmol/liter) did not differ significantly between case and control subjects [15.9 (SD, 15.2) versus 16.9 (SD, 14.9), respectively (P = 0.42)]. Odds ratios for prostate cancer risk estimated by conditional logistic regression for increasing quartiles of enterolactone concentration were 1.00 (referent), 0.72 [95% confidence interval (CI), 0.43-1.23], 0.98 (95% CI, 0.58-1.68), and 0.71 (95% CI, 0.42-1.21). Our findings do not support the hypothesis that enterolactone is involved in the development of prostate cancer.