Exposure of confluent human synovial McCoy's cells to near-freezing temperatures followed by rewarming at 37 degrees C resulted in endonuclease activation and cell death characteristic of a suicide process known as apoptosis. Both DNA fragmentation and cell killing were dependent on a sustained increase in the cytosolic Ca2+ concentration. Sensitivity to cold shock-induced endonuclease activation was critically dependent on the cell cycle (proliferative) status and limited to confluent cells, whereas cells in the logarithmic growth phase were completely resistant. However, DNA fragmentation was promoted in the proliferating McCoy's cells pretreated with H-7 or sphingosine, inhibitors of protein kinase C. In addition, phorbol ester, known to activate PKC, inhibited DNA fragmentation in the confluent cells. Our findings indicate that cold shock-induced DNA fragmentation in McCoy's cells is dependent on a sustained Ca2+ increase, and sensitivity to the process appears to be regulated by the status of protein kinase C.