A number of susceptibility loci for Alzheimer's disease (AD) have been identified including a region on Chromosome 10q21-22. Within this region the plasminogen activator urokinase gene (PLAU) was considered as a reasonable candidate from its functional implication in plasmin generation, a serine protease capable of degrading beta-Amyloid (Aβ) protein. We screened 56 single nucleotide polymorphisms (SNPs) around PLAU using 1751 individuals from four independent case-control samples (Munich, N = 679; Bonn N = 282; Brescia (Italy) N = 219; Perth (Australia) N = 557 and one discordant sib-pair sample (Munich N = 622). In brain tissue samples of neuropathologically confirmed cases with AD (N = 33) we analyzed plaque counts according to the risk allele. We identified that one functional exonic SNP (rs2227564) is associated with development of AD using the four independent case-control samples (Munich, P = 0.02; Bonn, P = 0.005; Brescia (Italy), P = 0.001; Perth (Australia), P = 0.03) and the discordant sib-pair sample (P = 0.001). In brain tissue, from neuropathologically confirmed cases with AD, we identified significantly higher plaque counts in carriers of the risk allele (N = 6; 60.3 ± 16.9) compared with non-carriers (N = 9; 26.3±8.8; P = 0.007). This study provides compelling evidence of a genetic and functional involvement of a common PLAU variant into the pathogenesis of AD. Further functional investigations are warranted to elucidate the specific role of PLAU, respectively, PLAU variants in the metabolism of Aβ proteins.