Data on genetic and environmental factors influencing PAI-1 levels and their consequent effect on clot lysis in black African populations are limited. We identified polymorphisms in the promoter area of the PAI-1 gene and determined their influence on PAI-1(act) levels and plasma clot lysis time (CLT). We also describe gene-environment interactions and the effect of urbanisation. Data from 2010 apparently healthy urban and rural black participants from the South African arm of the PURE study were cross-sectionally analysed. The 5G allele frequency of the 4G/5G polymorphism was 0.85. PAI-1(act) increased across genotypes in the urban subgroup (p = 0.009) but not significantly in the rural subgroup, while CLT did not differ across genotypes. Significant interaction terms were found between the 4G/5G polymorphism and BMI, waist circumference and triglycerides in determining PAI-1(act), and between the 4G/5G polymorphism and fibrinogen and fibrinogen gamma prime in determining CLT. The C428T and G429A polymorphisms did not show direct relationships with PAI-1(act) or CLT but they did influence the association of other environmental factors with PAI-1(act) and CLT. Several of these interactions differed significantly between rural and urban subgroups, particularly in individuals harbouring the mutant alleles. In conclusion, although the 4G/5G polymorphism significantly affected PAI-1(act), it contributed less than 1% to the PAI-1(act) variance. (Central) obesity was the biggest contributor to PAI-1(act) variance (12.5%). Urbanisation significantly influenced the effect of the 4G/5G polymorphism on PAI-1(act) as well as gene-environment interactions for the C428T and G429A genotypes in determining PAI-1(act) and CLT.