Thyroid hormone is essential for normal skeletal development. Hypothyroidism is associated with growth arrest, failure of chondrocyte differentiation and abnormal matrix synthesis. Thyroid hormone modulates the Indian hedgehog (Ihh)/parathyroid hormone-related peptide (PTHrP) feedback loop and regulates fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling. As heparan sulfate proteoglycans (HSPGs) are absolutely required by these signaling pathways we have investigated whether thyroid status effects HSPG expression within the growth plate. Tibial growth plate sections were obtained from 12 week old rats rendered euthyroid, thyrotoxic or hypothyroid at 6 weeks of age, 14 day old congenitally hypothyroid Pax8 null mice and TR/TR double null mice lacking all thyroid hormone receptors. Heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycan expression was determined by immunohistochemistry using three monoclonal antibodies. There was increased HS staining in growth plates from hypothyroid animals predominantly within the extracellular matrix of reserve and proliferative zones. Cellular HS staining was also increased particularly in pre-hypertrophic chondrocytes. T₃ regulation of HSPG core protein and HS synthetic and modification enzyme expression was studied in ATDC5 cells using semi-quantitative RT-PCR. Thyroid hormone negatively regulated expression of the core protein Gpc6, the polymerase Ext1 and the modification enzyme hs6st2. These studies demonstrate that the expression and distribution of growth plate proteoglycans is regulated by thyroid hormone and the regulation of HSPG expression provides an important additional link between FGF and Ihh signaling and T₃. These novel observations suggest that the cartilage matrix and especially HSPGs are critical mediators of the skeletal response to thyroid hormone.