The activity of PAK family kinases is regulated through interaction with the small GTPases Cdc42 and Rac1. PAKs are activated by the GTP-bound form of Cdc-42 and Rac1, and recruitment of PAKs to focal complexes has been implicated in Cdc42- and Rac1-dependent regulation of focal contact formation. PAK-interacting exchange factor (PIX) was identified in a screen for proteins that bind PAKs. Two forms of PIX have been identified: an 85 kDa protein designated αPIX and a 78 kDa protein designated βPIX. These proteins have 80% identity in their overlapping regions, which include myosin-like, pleckstrin (PH), Dbl (DH), and SH3 domains. In addition, αPIX contains a calponin-like domain at the N-terminus. The expression of βPIX is ubiquitous, while αPIX is expressed in heart, muscle, and thymus. PIX can act as a guanine nuleotide exchange factor for Rac1 and co-transfection of βPIX, Cdc42, and αPAK results in increased αPAK activity. PIX binding to PAK is required for localization of PAKs to focal complexes and injection of βPIX leads to Rac1-dependent membrane ruffling. Thus, PIX is important for PAK localization and activity during small GTPase-dependent regulation of cell morphology.