Abstract: Hyaluronan (HA), an extracellular linear polysaccharide of alternating N-acetyl-glucosamine and glucuronic acid residues, is ubiquitously expressed in vertebrates, where it affects a broad spectrum of physiological processes, including cell adhesion, migration and differentiation. The HA polymer is synthesized on the cytosolic side of the cell membrane by the membrane-embedded hyaluronan synthase (HAS). However, the process by which the extremely hydrophilic HA polymer is translocated across the membrane is unknown to date. The bacterial HAS from Streptococcus equisimilis (Se) shares a similar transmembrane topology and significant sequence identity with human HASs and likely synthesizes HA by the same mechanism. We demonstrate that the Se-HAS is both necessary and sufficient to translocate HA in a reaction that is tightly coupled to HA elongation. The purified Se-HAS is reconstituted into proteoliposomes (PLs) where it synthesizes and translocates HA. In vitro synthesized, high-molecular-weight HA remains tightly associated with the intact PLs in sedimentation experiments. Most importantly, the newly formed HA is protected from enzymatic degradation by hyaluronidase unless the PLs are solubilized with detergent, thereby demonstrating that HA is translocated into the lumen of the vesicle. In addition, we show that HA synthesis and translocation are spatially coupled events, which allow HA synthesis even in the presence of a large excess of HA-degrading enzyme. The coupled synthesis and membrane translocation of a biopolymer represents a novel membrane translocation mechanism and is likely applicable to the synthesis of some of the most abundant biopolymers, including chitin and cellulose. [Copyright &y& Elsevier]