Clinically relevant mature cartilage cells (chondrocytes) present challenges for use in cartilage tissue engineering applications, given their low capacity for cell division and tissue production. Since the in situ environment of chondrocytes is hypertonic relative to standard culture medium conditions, in this study we tested the hypothesis that using culture medium of a hypertonic, more physiologic osmolarity during both two-dimensional (2D) expansion of mature bovine chondrocytes (MBCs) and their subsequent encapsulation culture in three-dimensional (3D) agarose hydrogel constructs produces improved engineered tissue construct mechanical and biochemical properties. Results demonstrate that 2D expansion of MBCs in hypertonic (NaCl) medium before encapsulation yielded improved construct mechanical properties. However, 3D encapsulation culture of cells in hypertonic (NaCl) medium yielded poorer construct mechanical properties. Osmolarity-related differences in construct biochemical content and organization may have contributed to differences in mechanical properties, as construct glycosaminoglycan content correlated moderately with construct mechanical properties, and construct collagen distribution varied between 3D osmotic culture groups. Results of this study suggest that application of hypertonic (NaCl) medium during 2D mature chondrocyte expansion, but not 3D encapsulated chondrocyte culture, may serve as a convenient and inexpensive method for improving mechanical properties of expanded cell-seeded constructs.