Glycosaminoglycans (GAGs) are critical for extracellular matrix (ECM) integrity in cartilage but mechanisms regulating their synthesis are not defined. UDP-glucose dehydrogenase (UGDH) catalyses UDP-glucose oxidation to UDP-glucuronic acid, an essential monosaccharide in many GAGs. Our previous studies in articular surface (AS) cells from embryonic joints have established pivotal roles for mitogen-activated protein kinases (MAPK) in synthesis of the unsulfated GAG, hyaluronan (HA). We investigated the functional significance of UGDH in GAG production and chondrogenesis, and determined roles for MEK-ERK and p38MAPK pathways in regulating UGDH expression and function. Inhibitors of MEK and p38MAPK reduced UGDH protein in AS cells. Treatment with TGF-β (archetypal growth factor) increased UGDH expression, sulfated (s)-GAG/HA release and pericellular matrix formation in a p38MAPK-dependent manner. Retroviral overexpression of UGDH augmented HA/sGAG release and pericellular matrix elaboration, which were blocked by inhibiting MEK but not p38MAPK. UGDH overexpression increased cartilage nodule size in bone marrow culture, promoted chondrogenesis in limb bud micromass culture and selectively suppressed medium HA levels and modified GAG sulfation, as assessed by FACE analysis. Our data provide evidence that: (i) TGF-β regulates UGDH expression via p38MAPK to modulate sGAG/HA secretion, (ii) MEK-ERK, but not p38MAPK facilitates UGDH-induced HA and sGAG release, and (iii) increased UGDH expression promotes chondrogenesis directly and differential modifies GAG levels and sulfation. These results indicate a more diverse role for UGDH in the support of selective GAG production than previously described. Factors regulating UGDH may provide novel candidates for restoring ECM integrity in degenerative cartilage diseases, such as osteoarthritis.Arthritis Research Campaign.