When expanded through passage, chondrocytes lose their ability to produce high-quality cartilaginous matrix. This study attempts to improve the properties of constructs formed with expanded chondrocytes through alterations in the expansion protocol and the ratio of primary to expanded chondrocytes used to form cartilage constructs. A chondrogenically tuned expansion protocol provided similar monolayer growth rates as those obtained using serum-containing medium and enhanced cartilaginous properties of resultant constructs. Various ratios of primary to chondrogenically expanded chondrocytes were then self-assembled to form neocartilage. Biochemical analysis showed that constructs formed with only expanded cells had twice the GAG per wet weight and collagen II/collagen I ratio compared to constructs formed with primary chondrocytes. Biomechanically, compressive properties of constructs formed with only passaged cells matched the instantaneous modulus and exceeded the relaxation modulus of constructs formed with only primary cells. These counterintuitive results show that, by applying proper expansion and three-dimensional culture techniques, the cartilage-forming potential of adult chondrocytes expanded through passage can be enhanced over that of primary cells.