The establishment of polarity is an essential step in epithelial morphogenesis. Polarity proteins promote an apical/basal axis, which, together with the assembly of apical adherens and tight junctions, directed vesicle transport and the reorganization of the actomyosin filament network, generate a stable epithelium. The regulation of these cellular activities is complex, but the Rho family GTPase Cdc42 (cell division cycle 42) is known to play a key role in the establishment of polarity from yeast to humans. Two Cdc42 target proteins, the kinase PAK4 [p21 protein (Cdc42/Rac)-activated kinase 4] and the scaffold partitioning defective (Par) 6B, are required to promote the assembly of apical junctions in human bronchial epithelial cells. We show in the present paper that PAK4 phosphorylates Par6B at Ser143 blocking its interaction with Cdc42. This provides a potential new mechanism for controlling the subcellular localization of Par6B and its interaction with other proteins.