Influenza A virus NS1 is a multifunctional protein, and in virus-infected cells NS1 modulates a number of host-cell processes by interacting with cellular factors. Here, we report that NS1 binds directly to p85beta, a regulatory subunit of phosphatidylinositol-3-kinase (PI3K), but not to the related p85alpha subunit. Activation of PI3K in influenza virus-infected cells depended on genome replication, and showed kinetics that correlated with NS1 expression. Additionally, it was found that expression of NS1 alone was sufficient to constitutively activate PI3K, causing the phosphorylation of a downstream mediator of PI3K signal transduction, Akt. Mutational analysis of a potential SH2-binding motif within NS1 indicated that the highly conserved tyrosine at residue 89 is important for both the interaction with p85beta, and the activation of PI3K. A mutant influenza virus (A/Udorn/72) expressing NS1 with the Y89F amino acid substitution exhibited a small-plaque phenotype, and grew more slowly in tissue culture than WT virus. These data suggest that activation of PI3K signaling in influenza A virus-infected cells is important for efficient virus replication.