Rapid production of cartilaginous extracellular matrix (ECM) is required for scale up of any articular cartilage tissue engineering approach. Although several different methods have been investigated to increase the rate of cartilaginous ECM synthesis (e.g. growth factor stimulation, mechanical loading, etc.), there is evidence to suggest that cell cycle synchronization increases rate of ECM deposition. The issue with primary articular chondrocytes (PACs) is that routine methods to synchronize cells within a particular phase of the cell cycle rely on the use of monolayer culture, which is known to elicit cellular de-differentiation. This required development of a novel method of synchronizing cells within the S phase of the cell cycle during cell isolation. The objective of this study was to test whether synchronizing PACs would improve deposition of cartilaginous ECM in a three-dimensional culture model. Findings suggested that cell cycle synchronization was a viable method of improving the rate of matrix deposition in PACs.