The 4G allele of the PAI-1 -675(4G/5G) insertion/deletion promoter polymorphism has been associated with elevated plasma levels of PAI-1 and an increased risk of myocardial infarction. However, this allele has also been associated with a reduced risk of ischaemic stroke. In the brain, PAI-1 is mainly produced by astrocytes, and can reduce the neurotoxic effects exerted by tissue-type plasminogen activator during pathophysiologic conditions. The aim of the present study was to investigate whether the PAI-1 -675(4G/5G) polymorphism and the linked -844A/G polymorphism affect transcriptional activity of the PAI-1 gene in human astrocytes. Haplotype chromatin immunoprecipitation (haploChIP) was used in order to quantify allele-specific promoter activity in heterozygous cells. Protein-DNA interactions were investigated by electrophoretic mobility shift assay (EMSA). A clear allele-specific difference in PAI-1 gene expression was observed in astrocytes, where the haplotype containing the 4G and the -844A alleles was associated with higher transcriptional activity compared to the 5G and -844G-containing haplotype. EMSA revealed an allele-specific binding of nuclear proteins to the 4G/5G site as well as to the -844A/G site. Supershift experiments identified specific binding of the transcription factors Elf-1 and Elk-1 to the -844G allele. The relative impact of the different sites on allele-specific PAI-1 promoter activity remains to be determined. We demonstrate that common polymorphisms within the PAI-1 promoter affect transcriptional activity of the PAI-1 gene in human astrocytes, thus providing a possible molecular genetic mechanism behind the association between PAI-1 promoter variants and ischaemic stroke.