Listeria monocytogenes is a bacterial pathogen that invades cultured nonphagocytic cells. inhibitors and a dominant negative mutation were used to demonstrate that efficient entry requires the phosphoinositide (PI) 9-kinase p85 alpha-p110. Infection with L. monocytogenes caused rapid increases in cellular amounts of PI(3,4)P-2 and PI(3,4,5)P-3, indicating that invading bacteria stimulated PI 3-kinase activity. This stimulation required the bacterial protein In1B, host cell tyrosine phosphorylation, and association of p85 alpha with one or more tyrosine-phosphorylated proteins. This role for PI 3-kinase in bacterial entry may have parallels in some endocytic events.