The rapid cold-hardening (RCH) response increases the cold tolerance of insects by protecting against non-freezing, cold-shock injury. Apoptosis, or programmed cell death, plays important roles in development and the elimination of sub-lethally damaged cells. Our objectives were to determine whether apoptosis plays a role in cold-shock injury and, if so, whether the RCH response protects against cold-induced apoptosis in Drosophila melanogaster. The present study confirmed that RCH increased the cold tolerance of the adults at the organismal level. No flies in the cold-shocked group survived direct exposure to 7 degrees C for 2 h, whereas significantly more flies in the RCH group survived exposure to 7 degrees C for 2 h after a 2-h exposure to 5 degrees C. We used a TUNEL assay to detect and quantify apoptotic cell death in five groups of flies including control, cold-shocked, RCH, heat-shocked (37.5 degrees C, 30 min), and frozen (20 degrees C, 24 h) and found that apoptosis was induced by cold shock, heat shock, and freezing. The RCH treatment significantly improved cell viability by 38% compared to the cold-shocked group. Cold shock-induced DNA fragmentation shown by electrophoresis provided further evidence for apoptosis. SDS-PAGE analysis revealed an RCH-specific protein band with molecular mass of approximately 150 kDa. Western-blotting revealed three proteins that play key roles in the apoptotic pathway: caspase-9-like (apoptotic initiator), caspase-3-like (apoptotic executioner) and Bcl-2 (anti-apoptotic protein). Consequently, the results of this study support the hypothesis that the RCH response protects against cold-shock-induced apoptosis.