Hereditary multiple exostoses, a dominantly inherited genetic disorder characterized by multiple cartilaginous tumors, is caused by mutations in members of the EXT gene family, EXT1 or EXT2. The proteins encoded by these genes, EXT1 and EXT2, are endoplasmic reticulum-localized type II transmembrane glycoproteins that possess or are tightly associated with glycosyltransferase activities involved in the polymerization of heparan sulfate. Here, by testing a cell line with a specific defect in EXT1 in in vivo and in vitro assays, we show that EXT2 does not harbor significant glycosyltransferase activity in the absence of EXT1. Instead, it appears that EXT1 and EXT2 form a hetero-oligomeric complex in vivo that leads to the accumulation of both proteins in the Golgi apparatus. Remarkably, the Golgi-localized EXT1͞EXT2 complex possesses substantially higher glycosyltransferase activity than EXT1 or EXT2 alone, which suggests that the complex represents the biologically relevant form of the enzyme(s). These findings provide a rationale to explain how inherited mutations in either of the two EXT genes can cause loss of activity, resulting in hereditary multiple exostoses. Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by the formation of cartilagecapped tumors (exostoses) that develop from the growth plate of endochondral bone (1). This condition can lead to skeletal abnormalities, short stature, and in some instances, malignant transformation from exostoses to chondrosarcomas (2, 3) or osteosarcomas (4, 5). Although genetic linkage analysis has identified three different loci for HME, EXT1 on 8q24.1, EXT2 on 11p11-13, and EXT3 on 19p (6-8), most HME cases have been attributed to missense or frameshift mutations in either EXT1 or EXT2 (9-15). EXT1 and EXT2 encode 746-and 718-aa proteins, respectively, that are expressed ubiquitously in human tissues (9, 16).Previous studies using epitope-tagged constructs have demonstrated that EXT1 is a predominantly endoplasmic reticulum (ER)-localized glycoprotein whose expression enhances the synthesis of cell surface heparan sulfate (HS) (17). HS chains are composed of alternating residues of D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc) joined by 134 linkages, and a recent study has shown that both EXT1 and EXT2 harbor GlcA transferase (GlcA-T) and GlcNAc transferase (GlcNAc-T) activities that catalyze the polymerization of HS (18). EXT1 and EXT2 are structurally similar to previously identified glycosyltransferases in that they are type II transmembrane proteins comprising an N-terminal cytoplasmic tail, a transmembrane domain, a stalk, and a large globular domain that is likely to harbor enzymatic activity (19). Moreover, a truncated active form of EXT2 is secreted from cells and can be isolated from serum (18), which is a fate common to other ER and Golgilocalized glycosyltransferases, including EXTL2, which is an EXT homolog shown to encode an ␣1,4-N-acetylhexosaminyltransferase (20). However, several important questions are...