Hyaluronan (HA), a functionally essential glycosaminoglycan in vertebrate tissues and a putative virulence factor in certain pathogenic bacteria, is an extended linear polymer composed of alternating units of glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc). Uncertainty regarding the mechanism of HA biosynthesis has included the directionality of chain elongation, i.e. whether addition of monosaccharide units occurs at the reducing or non-reducing terminus of nascent chains. We have investigated this problem using yeast-derived recombinant HA synthases from Xenopus laevis (xlHAS1) and from Streptococcus pyogenes (spHAS). The enzymes were incubated with UDP-[3H]GlcUA and UDP-[14C]GlcNAc, under experimental conditions designed to yield HA chains with differentially labeled reducing-terminal and non-reducing terminal domains. Digestion of the products with a mixture of beta-glucuronidase and beta-N-acetylglucosaminidase exoenzymes resulted in truncation of the HA chain strictly from the non-reducing end and release of labeled monosaccharides. The change in 3H/14C ratio of the monosaccharide fraction, during the course of exoglycosidase digestion, was interpreted to indicate whether sugar units had been added at the reducing or non-reducing end. The results demonstrate that the vertebrate xlHAS1 and the bacterial spHAS extend HA in opposite directions. Chain elongation catalyzed by xlHAS1 occurs at the non-reducing end of the HA chain, whereas elongation catalyzed by spHAS occurs at the reducing end. The spHAS is the first glycosyltransferase that has been unanimously demonstrated to function at the reducing end of a growing glycosaminoglycan chain.