Human von Willebrand factor (VWF) gene sequences -487 to +247 function as an endothelial-specific promoter in vitro. Analysis of the activation mechanism of the VWF promoter has resulted in the identification of a number of cis-acting elements and trans-acting factors that regulate its activity. The GATA and Ets transcription factors were shown to function as activators of transcription, whereas NF1 and Oct1 were shown to repress transcription. We have reported the presence of another repressor element in exon 1 that interacted with a protein complex designated "R." In the absence of NF1 binding, inhibition of this interaction resulted in promoter activation in nonendothelial cells. We have now identified the "R" protein complex as the NFY transcription factor. Using DNA methylation interference assay and base substitution mutation analysis, we show that NFY interacts with a novel DNA sequence corresponding to nucleotides +226 to +234 in the VWF promoter that does not conform to the consensus NFY binding sequence CCAAT. The VWF gene does contain a CCAAT element that is located downstream of the TATA box and we show that the NFY factor also interacts with this CCAAT element. Using antibodies specific against the A, B, and C subunits of NFY, we demonstrate that the NFY complexes interacting with the CCAAT sequence have a composition similar to that of the repressor binding to the first exon sequences. The results of mutation analysis and transfection studies demonstrated that the interaction of NFY with the upstream CCAAT element is required for VWF promoter activation. Based on these results, we hypothesize that NFY can function both as a repressor and activator of transcription and its function may be modulated through its DNA binding sequences.