Signal Recognition Particle (SRP) mediates the delivery of roughly 30% of the newly-synthesized proteome to the eukaryotic endoplasmic reticulum or the bacterial plasma membrane. As such, it has served as a paradigm to understand the molecular basis of protein localization in all cells. At the core of this pathway are two homologous GTPases in the SRP and SRP receptor, which undergo coordinated conformational changes in their GTPase cycle to deliver translating ribosomes to the target membrane. Using biochemical reconstitution and single molecule fluorescence tools, we are elucidating the mechanism of mammalian SRP to understand the principle of accurate protein localization in higher eukaryotic cells and to unravel new layers of control and regulation of this essential protein targeting machine.