AbstractLigand binding to a given G protein-coupled receptor (GPCR) can modulate different signalling pathways downstream of the receptor depending on several factors, including the cell type, and more recently, the specific subcellular compartment in question. The concept of biased signalling not only challenges the conventional definition of agonism but also promises important potential therapeutic implications. By selectively targeting some pathways over others, biased ligands could in principle be designed to provide therapeutic benefit with fewer adverse effects. A major unanswered question, however, is whether GPCR ligands can be designed to modulate pools of receptors in distinct subcellular sites. To understand compartment-specific signalling, we have recently adapted Förster resonance energy transfer (FRET)-based signalling biosensors for in single cells. We have also been developing caged ligands designed to specifically target internal pools of receptors. The combination of the tool sets and new ligands allows us to answer some fundamental questions about where in the cell GPCRs act.