Osteoarthritis is the end result of damage to articular cartilage, which lacks the ability to self-repair. Tissue engineering of cartilage is a promising field of study that aims to promote healing of cartilage in vivo by manipulation of the chondrocytes that maintain the tissue, or through in vitro production of new cartilage for implantation into cartilage defects. Tissue-engineered cartilage constructs require mechanical stimulation to produce matrix components in quantities and proportions similar to native cartilage tissue, and adenosine triphosphate (ATP) is thought to be an autocrine/paracrine biochemical mediator of these mechanical forces on the cell, after its release from chondrocytes under mechanical stress. This study determined culture conditions for chondrocytes in 3D agarose scaffolds from mature donors undergoing total joint arthroplasty for the treatment of osteoarthritis, then supplemented these cells in vitro with exogenous ATP in concentrations varying from 50 nM to 1 mM in the presence of the radioisotopes [35S] and [3H]-proline, with radioisotope incorporation acting as markers of proteoglycan and collagen synthesis respectively. The basal concentrations of ATP in the chondrocyte cultures as well as the ATP half-life in the cultures were determined by lucifer/luciferase assay and luminometry. The P2Y receptor expression on the populations of chondrocytes from 8 donors was determined by flow cytometry, with largely varied individual expression and heterogeneity of P2Y1 and P2Y2 receptors. Exogenous ATP was found to increase synthesis of matrix components by 200% of the control cultures at doses of 100 nM to 1 µM. Patients with worse arthritis patterns, who were on chronic narcotic medications and who smoked were more likely to have a negative response to the exogenous ATP supplementation. The basal concentration of ATP in the cultures was less than 1 nM, and the ATP half-life varied from 1-2 hours, depending on the expression of P2Y1 receptors expressed by the donor’s chondrocyte population (R² = 0.99). Supplementation of exogenous ATP to tissue-engineered cartilage in vitro appears to be a promising technique for improving the matrix synthesis of these constructs.