Phosphatidylserine (PS), a phospholipid with a negatively charged head group, is an important constituent of eukaryotic membranes. Rather than being a passive component of cellular membranes, PS plays an important role in a number of signaling pathways. Signaling is mediated by proteins that are recruited and/or activated by PS in one of two ways: via domains that stereospecifically recognize the head group, or by electrostatic interactions with membranes that are rich in PS and therefore display negative surface charge. Such interactions are key to both intracellular and extracellular signaling cascades. PS, exposed extracellularly, is instrumental in triggering blood clotting and also serves as an "eat me" signal for the clearance of apoptotic cells. Inside the cell, a number of pathways depend of PS; these include kinases, small GTPases and fusogenic proteins. This review will discuss the generation and distribution of PS, current methods of phospholipid visualization within live cells, as well as the current understanding of the role of PS in both extracellular and intracellular signaling events.