Chondrocytes within articular cartilage respond to mechanical loading by adjusting the synthesis of extracellular matrix components such as collagen type II and the proteoglycan, aggrecan. This occurs via a mechanotransduction pathway which involves the release of ATP and activation of Ca2+ signalling [1,2]. Chondrocytes possess primary cilia, which in other cell types function as mechanoreceptors [3]. This study tests the hypothesis that chondrocyte primary cilia are required for mechanosensitive ATP release and up-regulation of matrix synthesis. Articular chondrocytes derived from Wild-type (WT) and Tg737 Oak Ridge Polycystic Kidney (ORPK) mice were cultured in 3D agarose constructs. Mutation of the Tg737 gene (IFT88) disrupts polaris expression, halting ciliogenesis. Cilia expression was studied by confocal immunofluorescence of acetylated α-tubulin. Cell-agarose constructs were subjected to mechanical loading at 1Hz and 0-15% compressive strain for 1 hour. ATP release was quantified by the luciferase assay and the expression of collagen II and aggrecan assessed by qPCR. To investigate proteoglycan production at a protein level, constructs were subjected to 24 hours of cyclic compression followed by quantification of sulphated glycosaminoglycan (sGAG) using a spectrophotometric biochemical assay. Primary cilia were present in WT chondrocytes but there was an almost complete absence in ORPK cells. Both cell types expressed collagen II and aggrecan genes and synthesised sGAG. However, in the unloaded state, ORPK cells exhibited increased aggrecan gene expression (p<0.0001, n=12) but reduced sGAG production (p<0.0001, n=36) compared to WT cells. There was no difference in the basal level of ATP secretion. Mechanical loading had no effect on viability or proliferation for either cell type. However for WT cells, loading increased ATP release (p<0.01, n=35), increased aggrecan gene expression (p<0.05, n=9) and increased sGAG production (p<0.05, n=36). By contrast, this mechanically induced proteoglycan synthesis was absent in ORPK cells, although these cells maintained mechanically-induced ATP secretion (p=0.001, n=35). Unpaired T-tests or Mann-Whitney U-tests were used as appropriate. To conclude, ORPK chondrocytes without cilia show disrupted basal proteoglycan production with a complete loss of mechanosensitive up-regulation of aggrecan expression and sGAG synthesis. However the loss of cilia did not influence the ATP response, indicating that cilia are involved in chondrocyte mechanotransduction downstream of mechanosensitive release of ATP. For example, this may involve Ca2+ dependent modulation of the P2 receptors. These experiments provide the first evidence that the primary cilium is central to chondrocyte matrix synthesis and mechanotransduction with fundamental implications for both healthy and arthritic tissue.