PI3Ks (phosphoinositide 3-kinases) regulate many critical cellular responses by producing PI(3,4,5)P(3) (phosphatidylinositol 3,4,5-trisphosphate). To facilitate the spatio-temporal characterization of PI(3,4,5)P(3) in living primary cells, we generated a novel strain of transgenic mice [AktPH (Akt pleckstrin homology domain)-GFP (green fluorescent protein) Tg (transgenic) mice] that express a fluorescent bioprobe for PI(3,4,5)P(3)/PI(3,4)P(2) (phosphatidylinositol 3,4-bisphosphate). By crossing AktPH-GFP Tg mice with strains of gene-targeted 'knockout' mice lacking a particular phosphoinositide-metabolizing enzyme, we have been able to evaluate the contribution of each enzyme to PI(3,4,5)P(3) localization in migrating neutrophils. Our results indicate that PI3Kgamma and the PI(3,4,5)P(3) phosphatase SHIP1 [SH2 (Src homology 2)-containing inositol phosphatase-1] are the key regulators of PI(3,4,5)P(3) dynamics during fMet-Leu-Phe (N-formylmethionyl-leucylphenylalanine; 'chemotactic peptide')-stimulated neutrophil migration. Our study has also validated the fluorescent transgenic strategy for studying PI(3,4,5)P(3) metabolism in physiological and pathological situations.