Much has been learned about the role of exofacial phosphatidylserine (PS) in apoptosis and blood clotting using annexin V. However, because annexins are impermeant and unable to bind PS at low calcium concentration, they are unsuitable for intracellular use. Thus little is known about the topology and dynamics of PS in the endomembranes of normal cells. We used two new probes—green fluorescent protein (GFP)–LactC2, a genetically encoded fluorescent PS biosensor, and 1-palmitoyl-2-(dipyrrometheneboron difluoride)undecanoyl-sn-glycero-3-phospho-l-serine (TopFluor-PS), a synthetic fluorescent PS analogue—to examine PS distribution and dynamics inside live cells. The mobility of PS was assessed by a combination of advanced optical methods, including single-particle tracking and fluorescence correlation spectroscopy. Our results reveal the existence of a sizable fraction of PS with limited mobility, with cortical actin contributing to the confinement of PS in the plasma membrane. We were also able to measure the dynamics of PS in endomembrane organelles. By targeting GFP-LactC2 to the secretory pathway, we detected the presence of PS in the luminal leaflet of the endoplasmic reticulum. Our data provide new insights into properties of PS inside cells and suggest mechanisms to account for the subcellular distribution and function of this phospholipid.