Several studies have reported that activation of G(q)-coupled receptors inhibits PI3K (phosphoinositide 3-kinase) signalling. In the present study, we used purified proteins to demonstrate that Galpha(q) directly inhibits p110alpha/p85alpha PI3K in a GTP-dependent manner. Activated Galpha(q) binds to the p110alpha/p85alpha PI3K with an apparent affinity that is seven times stronger than that for Galpha(q).GDP as measured by fluorescence spectroscopy. In contrast, Galpha(q) did not bind to the p110gamma PI3K. Fluorescence spectroscopy experiments also showed that Galpha(q) competes with Ras, a PI3K activator, for binding to p110alpha/p85alpha. Interestingly, co-precipitation studies using deletion mutants showed that Galpha(q) binds to the p85-binding domain of p110alpha and not to the Ras-binding domain. Expression of constitutively active Galpha(q)Q209L in cells inhibited Ras activation of the PI3K/Akt pathway but had no effect on Ras/Raf/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] signalling. These results suggest that activation of G(q)-coupled receptors leads to increased binding of Galpha(q).GTP to some isoforms of PI3K, which might explain why these receptors inhibit this signalling pathway in certain cell types.