Flow-mediated dilation (FMD) is the standard tool used to assess endothelial function. The premise behind the standard FMD test is that it serves as an endothelial-dependant nitric oxide bioassay; however, the endothelium may release additional dilatory molecules which contribute to FMD, most notably prostacyclin and endothelial-derived hyperpolarizing factor. The relative importance of these molecules to the dilatory response may vary substantially among individuals, particularly in response to a number of diseased states. This review discusses how each of these molecules may contribute to vasodilation, and considers the circumstances in which they may vary.