The larva of the drosophilid fly, Chymomyza costata, is probably the most complex metazoan organism that can survive submergence in liquid nitrogen (−196°C) in a fully hydrated state. We examined the associations between the physiological and biochemical parameters of differently acclimated larvae and their freeze tolerance. Entering diapause is an essential and sufficient prerequisite for attaining high levels of survival in liquid nitrogen (23% survival to adult stage), although cold acclimation further improves this capacity (62% survival). Profiling of 61 different metabolites identified proline as a prominent compound whose concentration increased from 20 to 147 mM during diapause transition and subsequent cold acclimation. This study provides direct evidence for the essential role of proline in high freeze tolerance. We increased the levels of proline in the larval tissues by feeding larvae proline-augmented diets and found that this simple treatment dramatically improved their freeze tolerance. Cell and tissue survival following exposure to liquid nitrogen was evident in proline-fed nondiapause larvae, and survival to adult stage increased from 0% to 36% in proline-fed diapause-destined larvae. A significant statistical correlation was found between the whole-body concentration of proline, either natural or artificial, and survival to the adult stage in liquid nitrogen for diapause larvae. Differential scanning calorimetry analysis suggested that high proline levels, in combination with a relatively low content of osmotically active water and freeze dehydration, increased the propensity of the remaining unfrozen water to undergo a glass-like transition (vitrification) and thus facilitated the prevention of cryoinjury.insect freeze tolerance | cold hardiness | glass transition | metabolomics T emperate and polar insects overwinter at subzero body temperatures (1-3), which makes them suitable models for studying the principles of survival in animal cells, tissues, and complex organisms at cryothermic conditions. The drosophilid fly, Chymomyza costata (Zetterstedt) (Diptera: Drosophilidae), appears to be a highly promising model. It is distributed over a cool-temperate, Holarctic area (4) and fully grown third instar larvae enter facultative winter diapause in response to long night length and/or low temperature (5). Diapause larvae supercool to between −15°C and −25°C (6) but can survive freezing to −100°C provided they undergo cold acclimation (1 mo at 4°C), are inoculated by external ice at −2°C, and are cooled at a slow rate of 0.1°C∕ min (7,8). Diapause, cold-acclimated larvae of C. costata were successfully cryopreserved at the temperature of liquid nitrogen (N 2 ) (−196°C) for 1 h (9). To our knowledge, larva of C. costata represents the most complex metazoan organism that has reportedly survived after submergence in liquid N 2 in a fully hydrated state. Physiological mechanisms underlying this unique capacity remain poorly understood. Inoculative freezing and a slow cooling rate allow time for osmoti...