Chondrocytes, the cellular component of the articular cartilage, have long been recognized as strain-sensitive cells, and have the ability to sense mechanical stimulation through surface receptors and intracellular signaling pathways. This stress-induced biological response of chondrocytes has been exploited to facilitate chondrocyte culture in in vitro systems and examples include the application of hydrostatic pressure, dynamic compression, hydrodynamic shear (i.e. rotating bioreactors) and application of low-intensity pulsed ultrasound (US). While the ability of US to influence chondrogenesis has been documented, the precise mechanisms of US induced stimulation continues to be investigated. As a consequence, there remains a critical need to evaluate the impact of US on chondrocytes in 3-D culture, which is a necessary microenvironment for maintaining the chondrocyte phenotype. In the present study an intermittent continuous ultrasound wave was employed to stimulate chondrocytes seeded in 3-D scaffolds. Non-US stimulated scaffolds served as the control. Both control and US stimulate groups were assayed for the total DNA content, morphology, and cartilage specific gene expression by RT-PCR. Our results show that chondrocytes when stimulated with continuous US for predetermined time intervals, possessed 1.2 to 1.4-times higher cellular viability than control and had higher levels of type-II collagen and aggrecan mRNA expression when compared to controls. Cell cytoskeleton structure was observed to be impacted by US and was reflected in actin, tubulin and vimentin structural distribution.