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1. Hatchlings of the North American Painted Turtle, Chrysemys picta (Schneider 1783) typically spend their first winter of life inside a shallow, terrestrial hibernaculum (the natal nest) where they commonly are exposed for extended periods to ice and cold. Current evidence indicates that turtles withstand such exposure by resisting freezing and becoming supercooled. 2. Supercooled hatchlings held at constant temperatures in the laboratory experience circulatory impairment and stagnant hypoxia, and consequently rely on anaerobic metabolism to meet a portion of their energy needs. As a result, lactate accumulates in bodies of supercooled animals. 3. The first experiment in the current investigation demonstrated that unfrozen hatchlings exposed to subzero temperatures like those recorded in a natural hibernaculum contained elevated quantities of lactic acid (and free glucose). This finding validates the widespread reliance on laboratory studies to gain insights regarding the physiology of animals overwintering in the field. 4. In a second study, hatchling Painted Turtles held for 20 days at −6 °C contained nearly twice as much lactate as turtles sampled after 10 days at that temperature. Hatchlings held for 10 days at −6 °C and then for another 10 days at −3 °C also contained more lactate than turtles sampled after 10 days at −6 °C, but not as much as the hatchlings that spent 20 days at −6 °C. Thus, animals held for part of the time at the higher subzero temperature still relied on anaerobic metabolism, but not to the same extent as turtles held continuously at the lower temperature. In contrast, hatchlings held for 10 days at −6 °C and then for another 10 days at either 0 °C or +3 °C contained no more lactate than control animals that never were exposed to subzero temperatures. Hatchlings exposed for the second 10 days to either 0 °C or +3 °C apparently were able to catabolize or otherwise process all the lactate that was accumulated during the first 10 days of their treatment. 5. Free glucose in bodies of hatchlings was elevated in all animals exposed to subzero temperatures, even when the initial exposure was followed by 10 days at temperatures as high as +3 °C. This finding has important implications with regard to the substrate that is used to support intermediary metabolism in supercooled turtles as well as to the metabolic pathways that are used to remove accumulated lactate once body temperature of the turtles rises at least to 0 °C. Functional Ecology (2005) doi: 10.1111/j.1365-2435.2005.01004.x [ABSTRACT FROM AUTHOR]