Phosphoinositide 3-kinase (PI3K), PTEN and localized phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] play key roles in chemotaxis, regulating cell motility by controlling the actin cytoskeleton in Dictyostelium and mammalian cells. PtdIns(3,4,5)P3, produced by PI3K, acts via diverse downstream signaling components, including the GTPase Rac, Arf-GTPases and the kinase Akt (PKB). It has become increasingly apparent, however, that chemotaxis results from an interplay between the PI3K-PTEN pathway and other parallel pathways in Dictyostelium and mammalian cells. In Dictyostelium, the phospholipase PLA2 acts in concert with PI3K to regulate chemotaxis, whereas phospholipase C (PLC) plays a supporting role in modulating PI3K activity. In adenocarcinoma cells, PLC and the actin regulator cofilin seem to provide the direction-sensing machinery, whereas PI3K might regulate motility.