Updated project metadata. G protein-coupled receptors (GPCRs) are the largest class of cell surface signaling proteins; they participate in all physiological processes and are the targets of 30% of marketed drugs. Typically, nanomolar-micromolar concentrations of ligand are used to activate GPCRs in experimental systems. However, by measuring cAMP with increased spatial and temporal resolution, we can now detect GPCR responses to an extraordinarily wide range of ligand concentrations: from attomolar to millimolar. Mathematical modeling shows that the addition of femtomolar concentrations of ligand can activate a significant proportion of cells provided that a cell can be activated by 1-2 binding events. In addition to cAMP, activation of the endogenous b2-adrenoceptor (b2AR) and muscarinic M3R by femtomolar concentrations of ligand in cell lines and human cardiac fibroblasts causes sustained increases in nuclear ERK or cytosolic PKC, respectively. These responses are spatially and temporally distinct from those that occur at higher concentrations of ligand, and result in a unique proteomic profile. This highly sensitive signaling is dependent on the GPCRs forming pre-assembled higher-order signaling complexes at the plasma membrane. Recognizing that GPCRs respond to ultra-low concentrations of neurotransmitters and hormones challenges established paradigms of drug action and provides a new dimension of GPCR activation that is quite distinct from that typically observed.