The lignan enterolactone produced by the intestinal microflora from dietary precursors has been hypothesized to protect against coronary heart disease. The present study examined the association between serum enterolactone concentration and the risk of coronary heart disease. A prospective case-cohort study was conducted among male smokers randomized to receive a placebo supplement in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (1986-1999). Serum enterolactone concentrations were measured by the gas chromatography-mass spectrometry method in serum collected at trial baseline from 340 men diagnosed with nonfatal myocardial infarction (n = 205) or coronary death (n = 135) during follow-up and from the randomly selected subcohort of 420 subjects. The classic risk factors-adjusted rate ratios for all coronary heart disease events in increasing quintiles of enterolactone were 1.00 (referent), 0.85 (95% confidence interval (CI): 0.51, 1.43), 0.59 (95% CI: 0.35, 1.00), 0.69 (95% CI: 0.40, 1.16), and 0.63 (95% CI: 0.33, 1.11), and the p(trend) was 0.07. For the highest versus the lowest quintile of enterolactone, the rate ratios for nonfatal myocardial infarction and coronary death were 0.67 (95% CI: 0.37, 1.23; p(trend) = 0.10) and 0.57 (95% CI: 0.26, 1.25; p(trend) = 0.18), respectively. In conclusion, only weak support for the association between serum enterolactone concentration and coronary heart disease was found.