Endothelial nitric oxide synthase (eNOS) is important for cardiovascular homeostasis, vessel remodeling, and angiogenesis. Given the impact of endothelium- derived nitric oxide (NO) in vascular biology, much work in the past several years has focused on the control of NO synthesis by regulatory proteins that influence its function. Indeed calcium-activated calmodulin is important for regulation of NOS activity. Herein we discuss why other proteins, in addition to calmodulin, are necessary for eNOS regulation and summarize the biology of negative and positive regulators of eNOS function in vitro, in cells, and in blood vessels.