BACKGROUND: The NOTCH4 gene is located at 6p21.3, a site shown in several studies to have significant linkage with Alzheimer's disease. OBJECTIVE: To investigate the potential impact of two polymorphisms within this gene on the risk of developing Alzheimer's disease. METHODS: Genotyping of promoter and 5'-UTR polymorphisms was done in Scottish, English, and French populations. The potential functionality of the 5'-UTR polymorphism was assessed by testing its impact on A beta load in Alzheimer brains and also by undertaking electrophoretic mobility shift assays and transfection experiments. RESULTS: No association of the Notch4 polymorphisms alone with the disease was observed in any of the populations. However, an interaction of the 5'-UTR C/T polymorphism with the epsilon 4 allele of the APOE gene was detected in United Kingdom populations but not in the French. No relation between the 5'-UTR polymorphism and A beta loads was detected overall or in the presence or absence of the epsilon 4 allele. No DNA protein specific binding was found with proteins from neuroblastoma, glioma, or astrocytoma cells, and no allele dependent transcriptional activity was detected. CONCLUSIONS: No association between two NOTCH4 polymorphisms alone and Alzheimer's disease was observed in the three populations, but there was evidence of an increased risk associated with the 5'-UTR CC genotype in epsilon 4 bearers in the United Kingdom. As no functionality for this polymorphism could be determined, it is likely that the interaction is spurious or results from a linkage disequilibrium of this 5'-UTR polymorphism with another marker elsewhere in the 6p21.3 locus.