Hereditary multiple exostoses (HME) is an autosomal dominant condition in which bony outgrowths occur from the juxtaepiphyseal regions of the long bones. In a few percent of cases these exostoses undergo malignant transformation to chondrosarcomas. HME results from mutations in one of two homologous genes, EXT1 and EXT2. These are members of a new gene family that is conserved from Caenorhabditis elegans to higher vertebrates. In humans this family comprises five genes which are most conserved at their C-termini, but they do not contain any discernible functional motifs and their function(s) is unclear. Indirect evidence suggests that EXT proteins are involved in glycosaminoglycan synthesis, act as tumor suppressors and affect hedgehog signaling. One recent study has also reported that these proteins co-purify with glycosyltransferase (GlcA and GlcNAc transferase) activity and on that basis it has been postulated that they are themselves glycosyl-transferases. We performed two-hybrid screens with a fragment of EXT2 from the region that is most highly conserved in the gene family and identified two interacting proteins: the tumor necrosis factor type 1 associated protein and a novel UDP-GalNAc:poly-peptide N -acetylgalactosaminyltransferase. Significantly, both these interactions were abrogated by a disease-causing EXT mutation, indicating that they are important in the etiology of HME. The EXT2-GalNAc-T5 interaction provides the first direct physical link between EXT proteins and known components of glycosamino-glycan synthesis.