Mechanical compression of cartilage is associated with a rise in the interstitial osmotic pressure, which can alter cell volume and activate volume recovery pathways. One of the early events implicated in regulatory volume changes and mechanotransduction is an increase of intracellular calcium ion ([Ca²⁺]_i). In this study, we tested the hypothesis that osmotic stress initiates intracellular Ca²⁺ signaling in chondrocytes. Using laser scanning microscopy and digital image processing, [Ca²⁺]_i and cell volume were monitored in chondrocytes exposed to hyper-osmotic solutions. Control experiments showed that exposure to hyper-osmotic solution caused significant decreases in cell volume as well as transient increases in [Ca²⁺]_i. The initial peak in [Ca²⁺]_i was generally followed by decaying oscillations. Pretreatment with gadolinium, a non-specific blocker of mechanosensitive ion channels, inhibited this [Ca²⁺]_i increase. Calcium-free media eliminated [Ca²⁺]_i increases in all cases. Pretreatment with U73122, thapsigargin, or heparin (blockers of the inositol phosphate pathway), or pertussis toxin (a blocker of G-proteins) significantly decreased the percentage of cells responding to osmotic stress and nearly abolished all oscillations. Cell volume decreased with hyper-osmotic stress and recovered towards baseline levels throughout the duration of the control experiments. The peak volume change with 550 mOsm osmotic stress, as well as the percent recovery of cell volume, was dependent on [Ca²⁺]_i. These findings indicate that osmotic stress causes significant volume change in chondrocytes and may activate an intracellular second messenger signal by inducing transient increases in [Ca²⁺]_i.