Objective: Mechanical stimulation is a widely used method to enhance the formation and properties of tissue-engineered cartilage. While this approach can be highly successful, it may be more efficient and effective to harness the known underlying mechanotransduction pathways responsible. With this aim, the purpose of this study was to assess the effect of directly stimulating the purinergic receptor pathway through exogenous adenosine 5'-triphosphate (ATP) in absence of externally applied forces. Methods: Isolated bovine articular chondrocytes were seeded in high density, 3D culture and supplemented with varying doses of ATP for up to 4 weeks. The effects on biosynthesis, extracellular matrix accumulation and mechanical properties were then evaluated. Experiments were also conducted to assess whether exogenous ATP elicited any undesirable effects, such as: inflammatory mediator release, matrix turn-over and mineralization. Results: Supplementation with ATP had a profound effect on the growth and maturation of the developed tissue. Exogenous ATP (62.5-250. μM) increased biosynthesis by 80-120%, and when stimulated for a period of 4 weeks resulted in increased matrix accumulation (80% increase in collagen and 60% increase in proteoglycans) and improved mechanical properties (6.5-fold increase in indentation modulus). While exogenous ATP did not stimulate the release of inflammatory mediators or induce mineralization, high doses of ATP (250μM) elicited a 2-fold increase in matrix metalloproteinase-13 expression suggesting the emergence of a catabolic response. Conclusions: Harnessing the ATP-purinergic receptor pathway is a highly effective approach to improve tissue formation and impart functional mechanical properties. However, the dose of ATP needs to be controlled as not to elicit a catabolic response. © 2010 Osteoarthritis Research Society International.