Mutations in the plasminogen activator inhibitor-1 (PAI-1) gene, along with altered PAI-1 and tissue-type plasminogen activator (tPA) levels, have been implicated in stroke pathogenesis. We investigated the association of PAI-1 and tPA levels with stroke as a function of PAI-1 4G/5G and -844G/A genotypes, as well as the link between these PAI-1 gene variants and stroke risk, in a case-control study of 135 ischemic stroke patient, diagnosed according to clinical and radiologic findings and confirmed by computed tomography scan. Controls (n = 118) were age- and sex-matched and had no personal/family history of stroke. PAI-1 4G/5G and -844G/A genotyping were done by polymerase chain reaction-restriction fragment length polymorphism, and PAI-1 and tPA levels were measured by enzyme immunoassay. Significant elevation in PAI-1 and marked reduction in tPA levels were seen in stroke patients and were correlated with 4G/5G, but not with -844G/A, PAI-1 variants. Whereas the frequencies of 4G or -844A alleles were comparable between patients and controls, 4G/4G carriers had reduced risk of stroke compared with other genotypes (odds ratio [OR] = 0.54; 95% confidence interval [CI] = 0.31-0.95). The 4G/-844A haplotype also was more closely associated with reduced stroke risk (OR = 0.43; 95% CI = 0.20-0.97) than 5G/-844A or 4G/-844G haplotypes. Regression analysis demonstrated that 4G homozygosity (OR = 0.176), hypertension (OR = 6.288), and body mass index (OR = 1.325) were independent predictors of stroke. The protective effect of 4G allele against stroke suggests involvement of PAI-1 4G/5G polymorphism in stroke through a mechanism not related to fibrinolysis, possibly involving altered plaque stabilization, and/or through antagonism of tPA effects.