The migration of smooth muscle cells is a critical event in the pathogenesis of vascular diseases. We have investigated the role of hyaluronan (HA) and the hyaluronan receptor RHAMM in the migration of adult bovine aortic smooth muscle cells (BASMC). Cultured BASMC migrated from the leading edge of a single scratch wound with increased velocity between 1 and 24 h. Polyclonal anti-RHAMM antisera that block HA binding with this receptor abolished smooth muscle cell migration following injury. HA stimulated the random locomotion of BASMC and its association with the cell monolayer increased following wounding injury. Immunoblot analysis of wounded monolayers demonstrated a novel RHAMM protein isoform that appeared within one hour after injury. At the time of increased cell motility after wounding, FACS analysis demonstrated an increase in the membrane localization in approximately 25% of the cell population. Confocal microscopy of injured monolayers confirmed that membrane expression of this receptor was limited to cells at the wound edge. Collectively, these data demonstrate that RHAMM is necessary for the migration of smooth muscle cells and that expression and distribution of this receptor is tightly regulated following wounding of BASMC monolayers.