In cartilage tissue engineering an optimized culture system, maintaining an appropriate extracellular environment (e.g., pH of media), can increase cell proliferation and extracellular matrix (ECM) accumulation. We have previously reported on a continuous-flow bioreactor that improves tissue growth by supplying the cells with a near infinite supply of medium. Previous studies have observed that acidic environments reduce ECM synthesis and chondrocyte proliferation. Hence, in this study we investigated the combined effects of a continuous culture system (bioreactor) together with additional buffering agents (e.g., sodium bicarbonate [NaHCO₃]) on cartilaginous tissue growth in vitro. Isolated bovine chondrocytes were grown in three-dimensional cultures, either in static conditions or in a continuous-flow bioreactor, in media with or without NaHCO₃. Tissue constructs cultivated in the bioreactor with NaHCO₃-supplemented media were characterized with significantly increased (p<0.05) ECM accumulation (glycosaminoglycans a 98-fold increase; collagen a 25-fold increase) and a 13-fold increase in cell proliferation, in comparison with static cultures. Additionally, constructs grown in the bioreactor with NaHCO₃-supplemented media were significantly thicker than all other constructs (p<0.05). Further, the chondrocytes from the primary construct expanded and synthesized ECM, forming a secondary construct without a separate expansion phase, with a diameter and thickness of 4 mm and 0.72 mm respectively. Tissue outgrowth was negligible in all other culturing conditions. Thus this study demonstrates the advantage of employing a continuous flow bioreactor coupled with NaHCO₃ supplemented media for articular cartilage tissue engineering.