Endothelial Differentiation-related Factor (EDF)-1 is a low molecular weight polypeptide downregulated in endothelial cells exposed to HIV-1-Tat or the phorbol ester TPA. EDF-1 acts in the cytosol as a calmodulin binding protein, and in the nucleus as a transcriptional coactivator. Here, we show that EDF-1 is downregulated in non-proliferating microvascular endothelial cells. Indeed, both quiescence and senescence reduce the levels of EDF-1 and this is due to protein degradation through the proteasome. We also describe a different subcellular localization of EDF-1 which is mainly nuclear in senescent 1G11 cells. Since (i) endothelial nitric oxide (NO) seems to play a role in endothelial proliferation and (ii) NO is an important mediator involved in the control of vascular tone, inflammatory responses and angiogenesis, it is noteworthy that senescence downregulates the expression and the activity of endothelial nitric oxide synthase (eNOS) in microvascular endothelial cells. On the contrary, quiescence does not affect NOS expression and activity. The modulation of EDF-1 in microvascular endothelial cells might offer new insights into the molecular events involved in angiogenesis and in microvascular dysfunctions in the elderly.