Phospholipase C-β proteins form a highly conserved enzyme family that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers inositol 1,4,5-triphosphate (IP3) and diacylglycerol, which mobilize intracellular calcium and stimulate the activity of protein kinase C. PLCβ isoforms are activated through direct interactions with heterotrimeric G proteins of the Gαq family, Gβγ heterodimers and small GTPases such as Rac1. PLCβ proteins have a highly conserved catalytic core comprised of an N-terminal pleckstrin homology (PH) domain, followed by four EF hand domains, a triose phosphate isomerase (TIM) barrel-like catalytic domain split into X and Y halves by a variable linker (XY-linker), and a C2 domain. In the basal state, the Hα2’ helix packs against a highly conserved, hydrophobic cleft formed between the TIM barrel and C2 domain of the catalytic core, in close proximity to the active site and X-Y linker. Since the intermolecular interaction of the Hα1-Hα2 module with Gαq and the intramolecular interaction of Hα2′ with the catalytic core of PLCβ3 are competitive, disruption of the Hα2′-catalytic core interface enhances the affinity of Gαq for PLCβ3. In addition, deletion of the XY-linker enhances basal activity of the enzyme. Upon activation, Gαq-GTP directly binds the helix-turn-helix of PLCβ and reorients it away from membranes to relieve this component of steric occlusion of the lipase. Gαq simultaneously positions the catalytic core of PLCβ at the membrane interface, leading to repulsion of the XY-linker and opening the active site for catalysis. Activators of PLCβ isozymes use the membrane as a conduit to allosterically enhance the phospholipase activity of these enzymes.