We hypothesized that the mechanically active environment present in rotating bioreactors mediates the effectiveness of three-dimensional (3D) scaffolds for cartilage tissue engineering. Cartilaginous constructs were engineered by using bovine calf chondrocytes in conjunction with two scaffold materials (SM) (benzylated hyaluronan and polyglycolic acid); three scaffold structures (SS) (sponge, non-woven mesh, and composite woven/non-woven mesh); and two culture systems (CS) (a bioreactor system and petri dishes). Construct size, composition [cells, glycosaminoglycans (GAG), total collagen, and type-specific collagen mRNA expression and protein levels], and mechanical function (compressive modulus) were assessed, and individual and interactive effects of model system parameters (SM, SS, CS, SMCS and SSCS) were demonstrated. The CS affected cell seeding (higher yields of more spatially uniform cells were obtained in bioreactor-grown than dish-grown 3-day constructs) and subsequently affected chondrogenesis (higher cell numbers, wet weights, wet weight GAG fractions, and collagen type II levels were obtained in bioreactor-grown than dish-grown 1-month constructs). In bioreactors, mesh-based scaffolds yielded 1-month constructs with lower type I collagen levels and four-fold higher compressive moduli than corresponding sponge-based scaffolds. The data imply that interactions between bioreactors and 3D tissue engineering scaffolds can be utilized to improve the structure, function, and molecular properties of in vitro-generated cartilage.