The PI3K (phosphoinositide 3-kinase) pathway plays a fundamental role in cell growth, survival and migration. Recent evidence has shown the key but distinct roles played by class 1 isoforms PI3Kα and PI3Kβ in tumorigenesis, and consequently isoform-selective inhibitors have been hotly pursued. To identify key structural parameters governing inhibitor binding and selectivity (or non-selectivity) we have adopted in vitro site-directed mutagenesis of PI3K as a central technique. A series of point mutants were constructed in two regions [Region 1 (p110α 852-859) and Region 2 (p110α 768-778)] of both isoforms where non-conserved residues are exchanged for alanine or the corresponding residue in the other isoform. We have identified specific non-conserved residues that govern binding for α-selective inhibitors PIK75 (Ser773 of PI3Kα) (1) and the chemically distinct A66-S (Gln859) (2). We have also identified a key role for Tyr778 of PI3Kβ in facilitating binding of the PI3Kβ-selective inhibitor TGX-221. Remarkably, this residue does not participate directly in binding but acts as a conformational latch, allowing the exposure of an adjacent cryptic binding site. The mutagenesis data helps us to scrutinize binding models from different sources, thus providing important structural information when crystal structures are limited. It has also facilitated the design in our labs of novel PI3Kβ-selective inhibitors that have derived selectivity through a unique mechanism.