Phosphoinositide 3-kinase gamma (PI3K gamma) has been implicated in a variety of cellular signaling processes. It is a multifunctional enzyme with lipid and protein kinase activity that also acts as a scaffold protein. Although it is well known that membrane recruitment is essential for the phosphorylation of phosphoinositides, the cellular localization of PI3K gamma as a protein kinase remains unclear. It has merely been described that PI3K gamma protein kinase activity leading to MAPK activation seems to be restricted to a cytosolic localization. Here, we demonstrate that a hybrid-PI3K gamma having protein kinase, but not lipid kinase activity shows a similar cellular distribution with a high membrane association and comparable liposome binding behavior to wild-type PI3K gamma. Binding of PI3K gamma to liposomes mimicking the natural plasma membrane slightly stimulates autophosphorylation of PI3K gamma. However, liposomes containing an unphysiologically high amount of PI inhibit autophosphorylation of PI3K gamma. Finally, PI3K gamma bound to membrane fragments does not show autophosphorylation which is possibly due to protein-protein-interactions at the plasma membrane. This indicates that not only MAPK activation, but PI3K gamma protein kinase activity in general is localized in the cytosol. (C) 2010 Elsevier Inc. All rights reserved.