Signals generated by the extracellular matrix promote cell survival. Here we show that chondrocytes detached from their native extracellular matrix (ECM) and plated without serum at low density on poly-L-lysine undergo significant cell death that is associated with the production of reactive oxygen species (ROS). No cell death or ROS production was observed when cells were plated on fibronectin under the same conditions. Cell death on poly-L-lysine could be completely inhibited with the addition of either antioxidants or inhibitors of specific protein kinase C (PKC) isoforms including PKC-I. PKC-I was noted to translocate from the cytosol to the particulate membrane after plating on poly-L-lysine and this translocation was inhibited by the addition of an antioxidant. Time-course analyses implicated endogenous ROS production as a secondary messenger leading to PKC-I activation and subsequent chondrocyte cell death. Cell survival on poly-L-lysine was significantly improved in the presence of oligomycin or DIDS which suggested ROS production was via complex V of the the electron transport chain of the mitochondria and release of ROS to the cytosol was via voltage-dependent anion channels. Taken together, these results represent a novel mechanism by which ROS can initiate cell death through the activation of PKC-I.