Many insects, including Drosophila, succumb to the physiological effects of chilling at temperatures well above those causing freezing. Low temperature causes a loss of extracellular ion and water homeostasis in such insects, and chill injuries accumulate. Using an integrative and comparative approach, we examined the role of ion and water balance in insect chilling susceptibility/ tolerance. The Malpighian tubules (MT), of chill susceptible Drosophila species lost [Na(+)] and [K(+)] selectivity at low temperatures, which contributed to a loss of Na(+) and water balance and a deleterious increase in extracellular [K(+)]. By contrast, the tubules of chill tolerant Drosophila species maintained their MT ion selectivity, maintained stable extracellular ion concentrations, and thereby avoided injury. The most tolerant species were able to modulate ion balance while in a cold-induced coma and this ongoing physiological acclimation process allowed some individuals of the tolerant species to recover from chill coma during low temperature exposure. Accordingly, differences in the ability to maintain homeostatic control of water and ion balance at low temperature may explain large parts of the wide intra- and interspecific variation in insect chilling tolerance.