Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5KI)-beta participates in establishing polarity during leukocyte chemotaxis. Its final 83 amino acids localize PIP5KIbeta to the uropod of chemotaxing neutrophils and T cells, and interact with ezrin-radixin-moesin (ERM) proteins and EBP50 (4.1-ERM-binding phosphoprotein 50), a scaffold protein with 2 PDZ (PSD-95, disc large, ZO-1) domains. The structural motifs at the PIP5KIbeta C terminus that confer signaling specificity are, nonetheless, unknown. We show that the last 4 residues of PIP5KIbeta constitute an atypical PDZ-binding motif, which steers PIP5KIbeta to the uropod by binding to both EBP50 PDZ domains. Molecular modeling and mutagenesis indicated that PDZ-binding motif is necessary for PIP5KIbeta localization and for chemoattractant-induced neutrophil polarization. Polarity in cells that express PIP5KIbeta mutants lacking the PDZ-binding motif was restored by overexpression of PIP5KIbeta, but not of PIP5KIgamma_i2, another isoform that localizes to the neutrophil uropod. Our results identify an isoform-specific PDZ-binding motif in PIP5KIbeta, which confers specificity for PIP5KIbeta signaling at the uropod during leukocyte chemotaxis.