Background: Matrix metalloproteinase-3 (MMP3) is implicated in the pathogenesis and progression of atherosclerotic lesions. Previous studies suggested that MMP3 expression is influenced by a polymorphism (known as the 5A/6A polymorphism) in the promoter of the MMP3 gene and that this polymorphism is located within a cis-element that interacts with the transcription factor NFκB. In the present study, we sought to investigate whether MMP3 and NFκB were colocalized in atherosclerotic lesions and whether NFκB had differential effects on the two alleles of the MMP3 5A/6A polymorphism. Methodology/Principal Findings: Immunohistochemical examination showed that MMP3 and both the NFκB p50 and p65 subunits were expressed abundantly in macrophages in atherosclerotic lesions and that MMP3 expression was co-localized with p50 and p65. Chromatin immunoprecipitation experiments showed interaction of p50 and p65 with the MMP3 promoter in macrophages, with greater binding to the 5A allele than to the 6A allele. Reporter gene assays in transiently transfected macrophages showed that the 5A allele had greater transcriptional activity than the 6A allele, and that this allele-specific effect was augmented when the cells were treated with the NFκB activator lipopolysaccharides or cotransfected with p50 and/or p65 expressing plasmids, but was reduced when the cells were treated with the NFκB inhibitor 6-Amino-4-(4-phenoxyphenylethylamino)-quinazoline or transfected with a dominant negative mutant of IκB kinase-β. Conclusion: These results corroborate an effect of the 5A/6A polymorphism on MMP3 transcription and indicate that NFκB has differential effects on the 5A and 6A alleles. © 2010 Souslova et al.