The functional consequences of increased renal cortical hyaluronan that is associated with both acute injury and progressive scarring are unclear. The aim of this study was to characterize hyaluronan synthase-2 (HAS2)-driven HA synthesis and determine its effect on renal proximal tubular epithelial cell (PTC) function, because this is known to be the inducible form of HA synthase in this cell type. Overexpression of HAS2 mRNA increased HA generation, which in the supernatant predominantly was HA of large molecular weight, whereas there was an increase in low molecular weight HA in cell-associated fractions. This was associated with increased expression of hyaluronidases, inhibition of HA cable formation concurrent with reduction in HA-dependent monocyte binding, and increased pericellular HA matrix. Overexpression of HAS2 led to enhanced cell migration. HA can be modified by the covalent attachment of heavy chains that are derived from the serum protein inter-α-inhibitor (IαI), a process that is known to be catalyzed by TNF-α-stimulated gene 6 (TSG-6; an inflammation-associated protein). Enhanced migration was abrogated by blocking antibodies to either IαI or TSG-6. Addition of recombinant full-length TSG-6 (TSG-6Q) or TSG-6Q_Y94F, a mutant variant with impaired HA binding, increased cell migration. Both of these proteins were able to mediate the covalent transfer of heavy chains, from IαI and pre-α-inhibitor, onto HA. Addition of the isolated TSG-6-Link module (Link_TSG-6), which binds HA but is unable to form covalent complexes with IαI/pre-α- inhibitor, had no effect on migration, suggesting that TSG-6-mediated formation of heavy chain-HA complexes is critical in the formation of a pericellular HA matrix. Copyright © 2006 by the American Society of Nephrology.