Recently, we reported that an Alu insertion polymorphism of the tissue-type plasminogen activator (t-PA) gene is associated with vascular t-PA release rates in man. In the current study we searched the t-PA gene for putative functional genetic variants in linkage disequilibrium (LD) with this polymorphism. Healthy individuals with different Alu genotypes and contrasting t-PA release rates were studied. Regulatory and coding regions of the t-PA gene were sequenced. Eight single-nucleotide polymorphisms (SNPs) were identified. Three of these were in significant LD with the Alu polymorphism and consequently associated with t-PA release rates; one in the far upstream enhancer, one in exon 6, and one in intron 10. The enhancer SNP resides within a GC box. Electrophoretic mobility shift assay (EMSA) revealed a reduced binding affinity of Sp1 to the T allele, which is the allele associated with a low t-PA release rate. Variations in exon 6 and intron 10 were silent and without apparent effect on splicing, respectively.