We report the isolation of a cDNA encoding the third putative hyaluronan synthase, HAS3. Partial cDNAs and genomic fragments of mouse Has3 were obtained using a degenerate polymerase chain reaction approach. Partial clones facilitated the isolation of genomic and cDNA clones representing the mouse Has3 open reading frame. The open reading frame of 554 amino acids predicted a protein of 63.3 kDa with multiple transmembrane domains and several consensus HA binding motifs. Sequence comparisons indicated that mouse Has3 is most closely related to Has2 (71% amino acid identity) and also related to Has1 (57% identity), Xenopus laevis DG42 (56% identity), and Streptococcus pyogenes HasA (28% identity). Isolation of a genomic fragment of human HAS3 indicated high conservation between mouse and human sequences, similar to those observed for HAS1 and HAS2. Expression of the mouse Has3 open reading frame in transfected COS-1 cells led to high levels of hyaluronan synthesis, as determined through a classical particle exclusion assay, and by in vitro HA synthase assays. These results suggest that there are three putative mammalian hyaluronan synthases encoded by three separate but related genes which comprise a mammalian hyaluronan synthase (HAS) gene family.