Thrombomodulin is an endothelial cell surface receptor that transforms the procoagulant thrombin into an anticoagulant. A mutation in the thrombomodulin gene is a potential risk factor for venous and arterial thrombosis. We screened a region within the coding sequence of the thrombomodulin gene by single-strand conformation polymorphism analysis (SSCP) in a pilot study of 104 patients with myocardial infarction and 104 age, sex and race matched controls. We identified a 127G to A mutation in the gene, which predicts an Ala25Thr substitution, in 2 out of 104 patients (1 man and 1 woman) with myocardial infarction but in no controls. We assessed the risk of myocardial infarction associated with the mutation in a larger "Study of Myocardial Infarctions Leiden" (SMILE). Among 560 men with a first myocardial infarction before the age of 70, 12 were carriers of the Ala25Thr substitution. In a control group of 646 men, frequency-matched for age, seven were carriers of the Ala25Thr substitution. The allelic frequencies were 1.07% among patients and 0.54% among controls suggesting risk associated with the mutation [odds ratio (OR) 2.0, 95% confidence interval (CI) 0.8-5.1]. In patients aged below 50, the predicted risk was almost seven times increased (OR 6.5, CI 0.8-54.2). In the presence of additional risk factors, such as smoking and a metabolic risk factor, the predicted risk increased to 9-fold (OR 8.8. CI 1.8-42.2) and 4-fold (OR 4.4, CI 0.9-21.3), respectively. While not conclusive, these results strongly suggest that the Ala25Thr substitution is a risk factor for myocardial infarction, especially in young men, and when in the presence of additional risk factors.