OBJECTIVES: The uPA-PAI system has been shown to play a role in the development of a more aggressive tumor phenotype. The PAI-1 promoter 4G/5G polymorphism, furthermore, regulates free plasma PAI-1 levels in patients with myocardial infarction. Our aim was to verify if the different polymorphisms in the PAI-1 promoter are also associated with alterations in the intracellular accumulation of uPA-PAI complexes in human breast cancer. METHODS: Accumulation of uPA-PAI complexes inside the tumor cells was determined by means of immunohistochemistry, as previously described by our own group, and two extremely different sets of tumors were chosen, one of them with strong uPA-PAI complex reactivity inside more than 50% of tumor cells, the other with no demonstrable reactivity at all. Finally, the 4G/5G polymorphism of the PAI-1 promoter was studied in all of them by means of DNA extraction, PCR amplification of the PAI promoter sequence, and restriction polymorphism typing. RESULTS: Absence of intracellular uPA-PAI complex accumulation was significantly associated with the prevalence of the 4G allele and, conversely, the presence of uPA-PAI complexes inside the tumor cells was significantly associated with 5G/5G homozygosity (logistic regression, p = 0.0128). Furthermore, none of the 7 5G/5G homozygous tumors showed histological grade 3, as did 6/21 tumors in the group where the 4G allele was present. In spite of the low case number, this association of the 5G/5G polymorphism with a less aggressive phenotype almost reached statistical significance (Spearman's correlation test, p = 0.118). CONCLUSIONS: The 4G/5G polymorphism of the PAI-1 promoter seems indeed to be associated with different rates of uPA-PAI complex internalization by breast cancer cells. Complex accumulation inside the tumor cells is significantly related to 5G/5G homozygosity, and this shows a trend towards an association with a less aggressive, better-differentiated tumor phenotype.