Pak (p21-activated kinase) serine/threonine kinases have been shown to mediate directional sensing of chemokine gradients. We hypothesized that Pak may also mediate chemokine-induced shape changes, to facilitate leucocyte chemotaxis through restrictive barriers, such as the extracellular matrix. A potent inhibitor, Pak(i), was characterized and used to probe the role of Pak-family kinases in SDF-1alpha (stromal-cell derived factor-1alpha/CXCL12)-induced chemotaxis in a T cell model. Pak(i) potently inhibited SDF-1alpha-induced Pak activation by a bivalent mechanism, as indicated by its complete inactivation upon point mutation of two binding sites, but partial inactivation upon mutation of either site alone. Importantly, Pak(i) was not toxic to cells over the time frame of our experiments, since it did not substantially affect cell surface expression of CXCR4 (CXC chemokine receptor 4) or integrins, cell cycle progression, or a number of ligand-induced responses. Pak(i) produced dose-dependent inhibition of SDF-1alpha-induced migration through rigid filters bearing small pores; but unexpectedly, did not substantially affect the magnitude or kinetics of chemotaxis through filters bearing larger pores. SDF-1alpha-induced Pak activation was partly dependent on PIX (Pak-interactive exchange factor); correspondingly, an allele of beta-PIX that cannot bind Pak inhibited SDF-1alpha-induced chemotaxis through small, but not large pores. By contrast, other key players in chemotaxis: G(i), PI3K (phosphoinositide 3-kinase), and the Rho-family G-proteins, Rac and Cdc42 (cell division cycle 42), were required for SDF-1alpha-induced migration regardless of the barrier pore-size. These studies have revealed a distinct branch of the SDF-1alpha signalling pathway, in which the Rac/Cdc42 effector, Pak, and its partner, PIX, specifically regulate the cellular events required for chemokine-induced migration through restrictive barriers.