The current models of eukaryotic plasma membrane organization separate the plasma membrane into different environments created by lipids and interactions between membrane proteins and the cytoskeleton, but characterization of their physical properties, such as their sizes, lifetimes, and the partitioning of membrane components into each environment, has not been accomplished. Single-molecule (fluorophore) tracking (SMT) experiments are well suited to the noninvasive study of membrane properties. In SMT experiments, the position of a single fluorescently labeled protein or lipid probe is followed optically as it moves within the membrane. If the motion of the probe is unhindered, then the spatial trajectory of the molecule will follow two-dimensional Brownian motion. If the probe encounters a structure that in some way inhibits its movement, then the probe’s trajectory will deviate from Brownian motion. It is likely that even if a certain type of lipid or protein partitions strongly into one environment, each individual lipid or protein will spend some fraction of its lifetime in the less favorable environment. Because SMT follows the motion of an individual probe over a large area (～10 ? 10 ?m2), transitions between environments can be observed directly by monitoring the path of each protein or lipid. Additionally, heterogeneity owing to multiple populations of molecules permanently residing in different states may be distinguished from a single population of molecules transitioning between different states. By judicious choice of label, such that the motion of the labeled protein or lipid is unaffected by the label itself, and through the use of probes with different affinities for each membrane environment, SMT measurements in principle can reveal the structure of the plasma membrane.