Ca²⁺ signaling forms part of a possible mechanotransduction pathway by which chondrocytes may alter their metabolism in response to mechanical loading. In this study, a well-characterized model system utilizing bovine articular chondrocytes embedded in 4% agarose constructs was used to investigate the effect of physiological mechanical compressive strain applied after 1 and 3 days in culture. The intracellular Ca²⁺ concentration was measured by use of the ratiometric Ca²⁺ indicator indo 1-AM and confocal microscopy. A positive Ca²⁺ response was defined as a percent increase in Ca²⁺ ratio above a preset threshold. A significantly greater percentage of cells exhibited a positive Ca²⁺ response in strained constructs compared with unstrained controls at both time points. In strained constructs, treatment with either Ga3+ or EGTA significantly reduced the number of positive Ca²⁺ responders compared with untreated controls. These results represent an important step in understanding the physiological role of intracellular Ca²⁺in chondrocytes under mechanical compression.