Recent evidence suggests a role for phosphatidylinositol (PI) 3‐kinase in various inflammatory responses. In this study, the consequences of LPS‐induced PI 3‐kinase activation on cytokine and chemokine expression and the intracellular mechanisms of inflammatory activation were examined in mouse macrophages. LPS stimulation induced a complex formation between PI 3‐kinase and myeloid differentiation factor 88 (MyD88), which was followed by an induction of IL‐1β, tumor necrosis factor‐α (TNF‐α) and macrophage inflammatory protein (MIP)‐2. The induction of IL‐1β, but not of MIP‐2 or TNF‐α, was blocked by the PI 3‐kinase inhibitors LY294002 and wortmannin. The nuclear factor‐κB (NF‐κB) inhibitor pyrrolidinedithiocarbamate (PDTC) blocked the induction of IL‐1β and TNF‐α, but had no effect on MIP‐2 expression. Inhibition of PI 3‐kinase decreased the LPS‐induced transcriptional activity of NF‐κB, but it had no effect on the nuclear DNA binding activity of NF‐κB. These findings suggest that, while NF‐κB nuclear localization and DNA binding are necessary, they are not sufficient for transcriptional activation of the IL‐1β gene in the absence of PI 3‐kinase activity. Taken together, our results demonstrate that activation of Toll‐like receptor (TLR)‐4 results in PI 3‐kinase‐MyD88 complex formation, and that PI 3‐kinase activity selectively leads to cytokine induction downstream of TLR4.