Chondrocyte adhesion to cartilage may play an important role in the repair of articular defects by maintaining cells in positions where their biosynthetic products can contribute to the repair process. The objective of this in vitro study was to determine the effect of the duration of seeding time on the ability of chondrocytes to resist detachment from cartilage when subjected to mechanical perturbation (fluid-induced shear stress). Suspensions of adult bovine articular chondrocytes were prepared from primary, high-density monolayer cultures and infused into a parallel-plate shear-flow chamber where they settled onto 50-microm-thick sections of bovine articular cartilage at a density of approximately 20,000 cells/cm2. The chondrocytes were seeded and allowed to attach to the cartilage surface for specific durations (5-40 minutes) in medium including 10% serum at 22 degrees C, after which the cells were exposed to fluid flow-induced shear stresses (6-90 Pa). The fraction of detached cells at each shear stress was calculated from microscopic images. Shear stress was applied for 1 minute because this length of time was sufficient to induce steady-state cell detachment. Increasing the duration of cell seeding led to a more firm attachment of chondrocytes to cartilage. After 9 minutes of seeding, 50% cell detachment was induced by gravitational force alone. After 40 minutes of seeding, 50% detachment required 26 Pa of shear stress. Extrapolation of the data to account for the effect of repeated applications of cell suspensions to an individual cartilage substrate indicated that for a freshly prepared cartilage section, 50% detachment was induced by gravity after 25 minutes of seeding and by 2.3 Pa of shear stress after 40 minutes of seeding. The increase in resistance to shear stress-induced cell detachment with increasing seeding duration suggests that it may be beneficial to allow chondrocytes to stabilize in the absence of applied load for some time after chondrocyte transplantation for cartilage repair in vivo.