In this study, we report that the sequential application of physiologic deformational loading after culturing with the growth factor TGF-beta3 (for 2-3 weeks) yields significantly stiffer chondrocyte-seeded agarose constructs than cultures in which deformational loading was applied during the initial 2-3 week TGF-beta3 exposure period. Using this culture protocol, engineered constructs were found to reach Young's modulus and GAG levels similar to that of native (parent) articular cartilage after only 42 days of culture. The present study extends the work on the mechanical preconditioning of engineered cartilage constructs to include transient supplementation with TGF-beta3 in a clinically-relevant, chemically-defined, serum-free media formulation.