Background: The polysaccharide hyaluronan (HA) is abundant in pancreatic cancer (PC) tissue and promotes pancreatic cancer cell (PCC) motility in vitro. However, it is controversial as to whether high-molecular-weight HA (HMW-HA) or low-molecular weight HA(LMW-HA) is present in the pancreatic cancer stroma and whether PCC or pancreatic stellate cell (PSC) in PC tissue produces HA. We thereby aim to characterize the molecular weight and source of HA in PC tissue that promotes cancer cell motility. Methods: We analyzed the expression of hyaluronan synthase 2 (HAS2) and the hydrolyzing enzyme hyaluronidase 1 (HYAL1) in PCC lines and pancreatic stellate cells (PSCs) using real-time PCR. HA production in the supernatant of PCC lines and PSCs and in PC tissues was quantitatively and qualitatively examined. Finally, we knocked down HYAL1 expression in one of the PCC line PANC-1 cells and analyzed the impact on cell migration. Results: HAS2 was abundantly expressed in activated PSCs (aPSCs) but less so in quiescent PSCs (qPSCs) and PCC lines. The baseline expression of HYAL1 did not differ among the cell types. The concentration of HMW-HA was higher in the supernatant of aPSCs than in that of PCC lines. Treatment with exogenous HMW-HA promoted PANC-1 cell motility. Knockdown of HYAL1 decreased HMW-HA-promoted PANC-1 cell migration, which was accompanied by a decrease in intracellular HA levels. Conclusion: aPSCs are an important source of stromal HMW-HA, which promotes PCC migration in an HYAL1-dependent manner in PC. (C) 2019 Elsevier Inc. All rights reserved.