Articular cartilage is an avascular tissue that does not respond adequately to injuries, leaving permanent chondral defects or fibrocartilage filled osteochondral defects that cannot bear physiological stress, eventually failing. Current articular cartilage tissue engineering methods employ homogeneously cell-seeded scaffolds that do not recreate the zonal structure or the biomechanical function of the native tissue. Thus, the goal of this study was to take the first steps in re-creating the zonal structure, and thus function, of articular cartilage by examining the effects of zone, passage, diffusion, seeding density, and mechanical stimuli on chondrocytes. A technique for isolating zonal chondrocytes was developed and verified. Chondrocytes from the superficial and growth zones were found to be phenotypically different, though this difference diminished rapidly in passage. A critical seeding density was found for the culture of chondrocyte constructs in bioreactors. From this finding, a novel self-assembling process was developed. The selfassembling process was shown to form articular cartilage constructs more than 1 mm thick with 1/3 the stiffness of native tissue after 12 weeks. Zonal chondrocytes cultured using the self-assembling process retained phenotypic differences. Fifth passage, dedifferentiated chondrocytes were shown to cease collagen type I expression when selfassembled. Lastly, the self-assembling process was shown to benefit from intermittent hydrostatic pressure stimulation. This study can serve as a launching point for a series of projects whose governing hypothesis is that articular cartilage can be regenerated by following a cell-based in vitro tissue engineering approach.