In the present study we investigated the role of CD98 in intestinal epithelial cell adhesion and spreading using the Electric Cell-Substrate Impedance Sensing technology. An anti beta-1 integrin function blocking antibody abrogated the attachment and spreading of human intestinal Caco2-BBE cells on laminin 1 (partner of beta-1 integrin). Caco2-BBE cells that over-expressed CD98 showed decreased attachment and spreading on laminin 1, when compared to Caco2-BBE cells transfected with vector only. The effect of over-expressed CD98 on cell adhesion and spreading was almost abolished when cells over-expressed the chimeras CD69-CD98 (cytoplasmic tail/transmembrane domain of CD98 replaced with the cytoplasmic tail/transmembrane domain of CD69), CD98-CD69 (extracellular loop of CD98 replaced with the extracellular loop of CD69), CD98 without its PDZ binding domain, or CD98 with a point mutation in its PDZ binding domain. Furthermore we demonstrated that not only over-expressed CD98, but also CD69-CD98, CD98-CD69 and CD98 with a point mutation in its PDZ binding domain still co-precipitate with beta-1 integrin in Caco2-BBE transfectants. Together these results suggest that the extracellular PDZ binding domain is necessary for the modulation of beta-1 integrin-dependent intestinal epithelial cell adhesion.