Homing of lymphocytes to tissues is a biologically important multistep process that involves selectin-dependent rolling, integrin-dependent adhesion and chemokine-directed chemotaxis. The actin cytoskeleton plays a central role in lymphocyte adhesion and motility. Wiskott-Aldrich syndrome protein (WASP), the product of the gene mutated in Wiskott-Aldrich syndrome, and its partner, the Wiskott-Aldrich syndrome protein-interacting protein (WIP), play important roles in actin re-organization in T lymphocytes. We used mice with disruption of the WASP and WIP genes to examine the role of WASP and WIP in T cell homing. T cell homing to spleen and lymph nodes in vivo was deficient in WASP-/- and WIP-/- mice and severely impaired in WASP-/-WIP-/- double knockout (DKO) mice. Deficiency of WASP, WIP or both did not interfere with selectin-dependent rolling or integrin-dependent adhesion of T cells in vitro. Chemotaxis to stromal cell-derived factor-1alpha (SDF-1alpha) in vitro was mildly reduced in T cells from WASP-/- mice. In contrast, it was significantly impaired in T cells from WIP-/- mice and severely reduced in T cells from DKO mice. Cellular F-actin increase following SDF-1alpha stimulation was normal in WASP-/- and WIP-/- T cells, but severely reduced in T cells from DKO mice. Actin re-organization and polarization in response to SDF-1alpha was abnormal in T cells from all knockout mice. Early biochemical events following SDF-1alpha stimulation that are important for chemotaxis and that included phosphorylation of Lck, cofilin, PAK1 and extracellular regulated kinase (Erk) and GTP loading of Rac-1 were examined in T cells from DKO mice and found to be normal. These results suggest that WASP and WIP are not essential for T lymphocyte rolling and adhesion, but play important and partially redundant roles in T cell chemotaxis in vitro and homing in vivo and function downstream of small GTPases.