Mechanical loading is an important regulator of chondrocytes; however, many of the mechanisms involved in chondrocyte mechanotransduction still remain unclear. Here, poly(ethylene glycol) (PEG) hydrogels are proposed as a model system to elucidate chondrocyte response due to cell deformation, which is controlled by gel crosslinking (ρ x ).Bovine articular chondrocytes (50×10 6 cells/mL) were encapsulated in gels with three ρ x s and subjected to static (15% strain) or dynamic (0.3Hz or 1Hz, 15% amplitude strain) loading for 48h. Cell deformation was examined by confocal microscopy. Cell response was assessed by total nitric oxide (NO) production, proteoglycan (PG) synthesis ( 35 SO 4 2− -incorporation) and cell proliferation (CP) ([ 3 H]-thymidine incorporation). Oxygen consumption was assessed using an oxygen biosensor.An increase in ρ x led to lower water contents, higher compressive moduli, and higher cell deformations. Chondrocyte response was dependent on both loading regime and ρ x . For example, under a static strain, NO was not affected, while CP and PG synthesis were inhibited in low ρ x and stimulated in high ρ x . Dynamic loading resulted in either no effect or an inhibitory effect on NO, CP, and PG synthesis. Overall, our results showed correlations between NO and CP and/or PG synthesis under static and dynamic (0.3Hz) loading. This finding was attributed to the hypoxic environment that resulted from the high cell-seeding density.This study demonstrates gel ρ x and loading condition influence NO, CP, and PG synthesis. Under a hypoxic environment and certain loading conditions, NO appears to have a positive effect on chondrocyte bioactivity.