The Rho-like GTPases Cdc42, Rac, and Rho play key roles in the regulation of the actin cytoskeleton and are implicated in transcriptional activation and cell transformation. We have previously identified the invasion-inducing Tiam1 gene, which encodes an activator of Rac. In fibroblasts, Tiam1 induces Rac-mediated membrane ruffling, which requires the N-terminal pleckstrin homology (PHn) domain. Here we show that this PHn domain is part of a protein interaction domain, which mediates membrane localization of Tiam1. After subcellular fractionation, up to 50% of Tiam1 is recovered in the Triton X-100-insoluble high speed pellet that contains small protein complexes. The regions in Tiam1 that are responsible for these protein interactions comprise the PHn domain, an adjacent putative coiled coil region (CC), and an additional flanking region (Ex). Deletions in each of these regions abolish membrane localization of Tiam1 and membrane ruffling, suggesting that they function cooperatively. Indeed, only polypeptides encompassing the PHn-CC-Ex region, and not the PHn-CC or the Ex region, localize at the membrane. These results indicate that the N-terminal PH domain is part of a larger functional Tiam1 domain that mediates protein complex formation and membrane localization of Tiam1.