CD44 has been implicated to play an important role in a diverse range of physiological processes, which involve cell-matrix recognition, cell-cell adhesion and cell motility. There is increasing evidence that the highly conserved intracellular domain of CD44 may be involved in influencing these activities. CD44 is phosphorylated in vivo on serine residue(s). In view of the importance that phosphorylation has been accorded in a multitude of cellular regulatory processes, we have investigated the role of phosphorylation in the control of CD44. In this report we identify the sites of human CD44 phosphorylation by mutating the three conserved cytoplasmic serine residues. We show that both Ser323 and Ser325, but not Ser316, are required for phosphorylation in vivo and demonstrate that this event is not stimulated by phorbol esters. Clonal MDCK cell lines expressing both the single and double CD44 phosphorylation mutants have been generated. These cell lines have been used to directly assess the role of phosphorylation on CD44 localization in polarized epithelial cells and its association with the cytoskeleton.