The inferior biomechanical properties of in vitro-formed tissue remain a significant obstacle in bioengineering articular cartilage tissue. We have previously shown that cyclic compression (30 minutes, 1 kPa, 1 Hz) of chondrocytes isolated from full-thickness cartilage can induce greater matrix synthesis, although articular cartilage is composed of different subpopulations of chondrocytes, and their individual contribution to enhanced tissue formation has not been fully characterized. This study examines the contribution of chondrocyte subpopulations to this response. Bovine articular chondrocytes were isolated from superficial to mid zones (SMZs) or deep zones (DZs), placed in three-dimensional culture, and subjected to cyclic compression. DZ chondrocytes on calcium polyphosphate substrates formed thicker tissue than those from SMZs. Compression increased matrix accumulation in SMZ chondrocytes while decreasing accumulation in DZ chondrocytes. The SMZ and DZ chondrocytes also differed in their type 1 membrane-bound matrix metalloproteinase (MMP) and MMP-13 expression, enzymes that play a crucial role in mediating the response to mechanical stimulation. In addition, the duration of the culture period was important in determining the DZ response, raising the possibility that matrix accumulation plays a role in the response to stimulation. Understanding the cellular response to mechanical stimulation during tissue formation will facilitate our understanding of tissue growth and allow for further optimization of cartilage tissue formation in vitro.