The process of chondrocyte mechanotransduction is poorly understood. However, recent studies suggest the involvement of a purinergic calcium signaling pathway although the mechanism of ATP release has not been identified. The present study tests the hypothesis that cyclic compression opens hemichannels thereby triggering the release of ATP into the extracellular milieu activating P2 receptors. The well-established chondrocyte-agarose model was utilized enabling chondrocytes to be subjected to a 40-min period of cyclic compression at 0-15% strain and 1 Hz. The opening of hemichannels was determined using Lucifer yellow (LY) incorporation and fluorescence microscopy, whereas the release of ATP into the surrounding media was quantified using the luciferin-luciferase assay. Results indicated that cyclic compression activated hemichannels such that the percentage of cells showing LY incorporation increased from 50 to 70%. This was associated with a sevenfold increase in the release of ATP. Both LY incorporation and ATP release in response to mechanical loading were blocked by the hemichannel inhibitor, flufenamic acid. Treatment with apyrase or P2 receptor antagonists, suramin or oxidated-ATP, did not prevent the mechanically induced response. In conclusion, mechanical loading triggers release of ATP via hemichannels. Hence, this study provides the first evidence of hemichannel involvement in chondrocyte mechanobiology.