Interleukin (IL)-10 overexpression inhibits joint inflammation, however the effect of high local concentrations of IL-10 on chondrocyte homeostasis remains unclear. The aim of this study was to determine the effects of IL-10 overexpression on cartilage matrix production in three-dimensional (3D) chondrocyte cultures. Human articular chondrocytes were transduced with adenoviral vectors alone (adv/empty) or by vectors either overexpressing enhanced green fluorescence protein (adv/EGFP) or human IL-10 (adv/hIL-10) before their transfer to a 3D culture system. Non-transduced chondrocytes were used as controls. The expression of IL-10 or EGFP was confirmed using ELISA or flow cytometry. Chondrocytes synthesis of collagen types II and I, aggrecan, fibronectin and β1-integrin was determined over a period of 14 days post transduction using flow cytometry or immunohistochemistry. adv/EGFP or adv/IL-10 transduced chondrocytes expressed EGFP or secreted IL-10 detectable over the 2 weeks culture period. No suppression of collagen type II, aggrecan or β1-integrin synthesis by IL-10 overexpression was found and the deposition of collagen type I and fi bronectin remained unaffected compared to the controls. IL-10 overexpression does not impair key features of chondrocytes differentiated phenotype (e.g. collagen type II and aggrecan expression) suggesting the potential use of IL-10 for gene therapeutic approaches in the joint.