OBJECTIVE: Hyaluronan (HA) is an important constituent of the extracellular matrix and is known to regulate cellular events through binding to CD44 and the receptor for HA-mediated motility (RHAMM). Here we investigated the role of these receptors and the signaling pathways involved in HA-mediated effects in arterial smooth muscle cells (ASMC). METHODS: Effects of high-molecular weight HA (1 to 5 mg/ml) were analyzed in cultured ASMC from rat aorta. RESULTS: HA promoted actin stress fiber and lamellipodia formation and dose-dependently induced ASMC migration without effect on proliferation. Pull-down assay of Rho protein activity indicated that HA activated RhoA and Rac. HA-induced ASMC migration was not affected by the RhoA inhibitor Tat-C3 (10 microg/ml), the Rho kinase inhibitor Y-27632 (10 microM) and blocking anti-CD44 antibody ,but was reduced by the non-selective Rho protein inhibitor simvastatin (10 microM), the Rac inhibitor LT-toxin (1 mug/ml), small interfering RNA (siRNA) targeting Rac and the phosphatidyl inositol 3-kinase (PI3K) inhibitor LY294002 (25 microM), which also blocked HA-induced Rac activation. CD44 knockdown by siRNA inhibited HA-mediated RhoA activation without effect on ASMC migration. In contrast, siRNA targeting RHAMM inhibited both HA-induced migration and Rac activation. CONCLUSIONS: High-molecular weight HA independently activates RhoA and Rac through CD44 and RHAMM, respectively. HA-induced migration depends exclusively on RHAMM-mediated PI3K-dependent Rac activation.