This study examined effects of varying magnitudes of intermittent hydrostatic pressure (IHP) applied for different times on chondrogenesis of adult human mesenchymal stem cells (hMSCs) in vitro. hMSCs were exposed to 0.1, 1, and 10 MPa of IHP at a frequency of 1 Hz for 4 h/day for 3, 7, and 14 days in the presence of transforming growth factor (TGF-beta3). Chondrogenesis was characterized by gene expression, macromolecule production, and extracellular matrix deposition. Exposure of hMSCs to 0.1 MPa of IHP increased SOX9 and aggrecan mRNA expression by 2.2- and 5.6-fold, respectively, whereas type II collagen mRNA expression responded maximally at 10 MPa. Production of sulfated glycosaminoglycan responded to IHP of 1 MPa and 10 MPa, whereas collagen levels increased only at 10 MPa. Morphologically, matrix condensation occurred with increased IHP, concomitant with collagen expression. This study demonstrated that different levels of IHP differentially modulate hMSC chondrogenesis in the presence of TGF-beta3. The data suggest that tissue engineering of articular cartilage through application or recruitment of hMSCs can be facilitated by mechanical stimulation.