Lymphocyte recruitment to sites of inflammation involves a bidirectional series of cues between the endothelial cell (EC) and the leukocyte that culminate in lymphocyte migration into the tissue. Remodeling of the EC F-actin cytoskeleton has been observed after leukocyte adhesion, but the signals to the EC remain poorly defined. We studied the dependence of peripheral blood lymphocyte (PBL) transendothelial migration (TEM) through an EC monolayer in vitro on EC phosphatidylinositol 3-kinase (PI3K) activity. Lymphocytes were perfused over cytokine-activated EC using a parallel-plate laminar flow chamber. Inhibition of EC PI3K activity using LY294002 or wortmannin decreased lymphocyte TEM (48 ± 6% or 34 ± 7% respectively vs. control; mean ± SEM; p<0.05). Similarly, EC knockdown of the p85alpha regulatory subunit of PI3 kinase decreased lymphocyte transmigration. Treatment of EC with jasplakinolide to inhibit EC F-actin remodeling also decreased lymphocyte TEM to 24 ± 10% vs. control (p<0.05). EC PI3K inhibition did not change the strength of lymphocyte adhesion to the EC or formation of the EC docking structure after ICAM-1 ligation, whereas this was inhibited by jasplakinolide treatment. A similar fraction of lymphocytes migrated on control or LY294002-treated EC and localized to interendothelial junctions. However lymphocytes failed to extend processes below the level of VE-cadherin on LY294002-treated EC. Together these observations indicate that EC PI3K activity and F-actin remodeling are required during lymphocyte diapedesis and identify a PI3K-dependent step following initial separation of the VE-cadherin barrier.