Eph/ephrin signaling is pivotal in prenatal angiogenesis. Although their role in these processes has been extensively studied the mechanisms controlling eph/ephrin expression is not fully understood. To better understand the transcriptional regulation of arteriogenesis we have cloned and characterized the promoter of ephrin-B2 and its regulatory elements. The transcription start site was identified using 5' RACE and through deletion studies a minimal promoter region was determined. This 180 bp region located just upstream of the transcription start site has a high GC content and contains a consensus TATA-box which proved to be vital for basal transcriptional activity. In addition, putative regulatory elements for five transcription factors were identified. Mutational destruction of individual binding sites and chromatin immunoprecipitation confirmed that Meis1, MAZ and NFY physically interacted with the region and regulated promoter activity.