In neutrophils, the phosphoinositide 3-kinase/Akt signaling cascade is involved in migration, degranulation, and O₂⁻ production. However, it is unclear whether the Akt kinase isoforms have distinct functions in neutrophil activation. Here we report functional differences between the 2 major Akt isoforms in neutrophil activation on the basis of studies in which we used individual Akt1 and Akt2 knockout mice. Akt2^−/− neutrophils exhibited decreased cell migration, granule enzyme release, and O₂⁻ production compared with wild-type and Akt1^−/− neutrophils. Surprisingly, Akt2 deficiency and pharmacologic inhibition of Akt also abrogated phorbol ester-induced O₂⁻ production, which was unaffected by treatment with the phosphoinositide 3-kinase inhibitor LY294002. The decreased O₂⁻ production in Akt2^−/− neutrophils was accompanied by reduced p47^phox phosphorylation and its membrane translocation, suggesting that Akt2 is important for the assembly of phagocyte nicotinamide adenine dinucleotide phosphate oxidase. In wild-type neutrophils, Akt2 but not Akt1 translocated to plasma membrane upon chemoattractant stimulation and to the leading edge in polarized neutrophils. In the absence of Akt2, chemoattractant-induced Akt protein phosphorylation was significantly reduced. These results demonstrate a predominant role of Akt2 in regulating neutrophil functions and provide evidence for differential activation of the 2 Akt isoforms in neutrophils.