An endothelial nitric oxide synthase gene (NOS3) polymorphism in exon 7 (G894T), resulting in Glu298Asp substitution at protein level, has been associated with myocardial infarction, hypertension and coronary atherosclerosis in some populations. This polymorphism is usually identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, the procedures described to date do not eliminate the possibility of misclassification and either require confirmation by DNA sequencing or are time-consuming. In this study, a PCR-RFLP procedure to detect the G894T polymorphism at the NOS3 was optimized by the introduction of a constitutive cleavage site in the amplification product. This cleavage site provides an internal control for enzymatic activity to avoid mistyping. The method was validated by the study of 35 white unrelated individuals with familial hypercholesterolemia and 70 controls. The frequency of the variant allele (T) was similar between both groups (27% vs. 22%, NS), and comparable to the frequency found in other white populations. However, future studies are necessary to confirm these data. In summary, the optimized procedure for detection of the G894T NOS3 polymorphism is rapid, simple, and does not require confirmatory tests. Using this method, we found no association between this polymorphism and familial hypercholesterolemia.