Significance Activated PI3K Delta Syndrome (APDS) is a primary immunodeficiency disease caused by activating mutations in phosphoinositide 3-kinases (PI3Kδ). Activating mutations in either the p110δ catalytic or the p85α regulatory subunit of PI3Kδ result in APDS. Mutations in p85α leading to APDS are surprising, as other p85α-activating mutations are oncogenic when bound to the PI3Kα isoform. Using hydrogen–deuterium exchange mass spectrometry, we determined the molecular mechanisms by which APDS mutations in p110δ or p85α activate PI3Kδ and reveal why the p85α APDS2 mutant primarily activates PI3Kδ. All APDS mutants are potently inhibited by the PI3Kδ-specific inhibitor idelalisib. Together, the biophysical and biochemical data reveal insights into PI3Kδ regulation and provide a possible therapeutic strategy for treating patients with APDS.