The main function of plasminogen activator inhibitor type 1 (PAI-1) is to decrease fibrinolysis, which leads to fibrin accumulation. An elevated plasma PAI-1 concentration has been identified as a risk factor for the development of myocardial infarction, and an association between 1 polymorphism of the PAI-1 promoter and plasma PAI-1 levels has been described. Our aim was to identify new polymorphisms in the PAI-1 gene and to further examine the relationship between PAI-1 genotypes and circulating PAI-1 levels. We report the presence of 4 new polymorphisms that were identified by non-isotopic single-strand conformational polymorphism analysis followed by sequencing. These polymorphisms were investigated in relation to PAI-1 levels in a sample of 256 healthy men, aged 50-59 years, from France and Northern Ireland. Two G/A substitutions were detected at positions -844 and +9785. The former is in strong positive linkage disequilibrium with the previously described 4G/5G polymorphism at position -675. Two polymorphisms in the 3' untranslated region were identified. One corresponds to a T/G substitution at position +11,053 and is in negative linkage disequilibrium with the G/A substitution (+9785). The other is a 9-nucleotide insertion/deletion located between nucleotides +11,320 and +11,345 in a threefold-repeated sequence. This polymorphism is in strong positive linkage disequilibrium with the G/A substitution (+9785). The overall heterozygosity provided by the 5 PAI-1 polymorphisms (including the 4 new variants and the 4G/5G polymorphism) was .77. No significant association was found between PAI activity and genotypes; furthermore, the well known associations between PAI activity and body mass index, serum triglycerides, or insulin were homogeneous according to PAI-1 genotypes.