Accumulating evidence indicates that Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a membrane-anchored matrix metalloproteinase regulator, plays crucial roles in mammalian development and tumor suppression. Its mechanisms of action at the single cell level, however, remain largely unknown. In mouse fibroblasts, RECK is abundant around the perinuclear region, membrane ruffles and cell surface. Cells lacking Reck show decreased spreading, ambiguous anterior-posterior (AP) polarity, and increased speed and decreased directional persistence in migration; these characteristics are also found in transformed fibroblasts and fibrosarcoma cells with low RECK expression. RECK-deficient cells fail to form discrete focal adhesions, have increased levels of GTP-bound Rac1 and Cdc42, and a marked decrease in the level of detyrosinated tubulin, a hallmark of stabilized microtubules. RECK-deficient cells also show elevated gelatinolytic activity and decreased fibronectin fibrils. The phenotype of RECK-deficient cells is largely suppressed when the cells are plated on fibronectin-coated substrates. These findings suggest that RECK regulates pericellular extracellular matrix degradation, thereby allowing the cells to form proper cell-substrate adhesions and to maintain AP polarity during migration; this mechanism is compromised in malignant cells.