AbstractThe adhesion molecule CD44 and the membrane‐type matrix metalloproteinase MT1‐MMP act coordinately in tumor cells to promote cell invasion through a yet unclear mechanism. We are interested in studying the interplay between CD44 and MT1‐MMP in carcinoma cells embedded in HA containing three‐dimensional collagen I matrices (3D HA‐Col I) by time‐lapse confocal microscopy imaging. Here we report the in vivo interaction between CD44 and MT1‐MMP, revealed by fluorescence resonance energy transfer (FRET) microscopy. MT1‐MMP interacts with CD44 preferentially at the trailing edge of the invading tumor cells during rear retraction and on membrane fragments released during the invasion process. A fluorescent biosensor designed to monitor the proteolytic processing of CD44 by live cell imaging demonstrates that cleavage of the CD44 extracellular domain is enriched in the retracting rear ends of invasive tumor cells. Invasion assays showed that MT1‐MMP mediates CD44‐dependent tumor‐cell invasion, whereas CD44 is not essential for MT1‐MMP‐mediated invasion of 3D HA‐Col I matrices. Together, our results support a role for MT1‐MMP in cell retraction during CD44‐mediated cell invasion. Cell. Motil. Cytoskelton 2008. © 2008 Wiley‐Liss, Inc.