Dendritic cells (DCs), antigen-presenting cells that are able to initiate primary immune responses and to establish immunological memory, are activated by exposure to bacterial lipopolysaccharides (LPS), which leads to cell swelling, triggering ROS formation and stimulating migration. The function of DCs is regulated by the phosphoinositide 3 (PI3) kinase pathway. On the other hand, PI3 kinase is an important regulator of diverse transporters including the Na(+)/H(+) exchanger (NHE). The present study was performed to elucidate the role of PI3 kinase in NHE activity, cell volume, ROS formation, and migration. To this end, DCs were isolated from murine bone marrow, cytosolic pH (pH(i)) determined utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein fluorescence, Na(+)/H(+) exchanger activity from the Na(+)-dependent realkalinization after an ammonium pulse, cell volume from forward scatter in fluorescence-activated cell sorter analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate fluorescence, and migration utilizing transwell migration assays. Exposure of DCs to LPS led within 4 h to a gradual cytosolic acidification paralleled by a transient time- and dose-dependent increase of Na(+)/H(+) exchanger activity, cell swelling, enhanced ROS production, and stimulation of migration. The PI3K inhibitors Wortmannin (1 μM) or LY294002 (10 μM) significantly blunted the effects of LPS on NHE activity, cell volume, ROS production, and migration. The present observations disclose a critical role of PI3K signaling in the regulation of DC function following exposure to LPS.