Although many studies of ectothermic vertebrates have documented compensatory changes in cold hardiness associated with changes of season, much less attention has been paid to adjustment of physiological functions and survival limits following more acute exposure to cold. We investigated the ability of hatchling painted turtles (Chrysemys picta) to increase cold hardiness in response to brief exposure to a subzero temperature. Winter-acclimated turtles were "cold conditioned" by chilling them in the supercooled (unfrozen) state to -7 degrees C over a few days before returning them to 4 degrees C. These turtles fared no better than control animals in resisting freezing when cooled in the presence or absence of ice and exogenous ice nuclei. Survival following tests of freeze tolerance (freezing for about 70 h; minimum body temperature, -3.75 degrees C) was nominally higher in cold-conditioned turtles than in controls (36% vs. 13%, respectively), although the difference was not statistically significant. Of the survivors, cold-conditioned turtles apparently recovered sooner. Turtles subjected to cold shock (supercooling to -13 degrees C for 24 h, followed by rewarming to 0 degrees C) were strongly affected by cold conditioning: all controls died, but 50% of cold-conditioned turtles survived. We investigated potential mechanisms underlying the response to cold conditioning by measuring changes in levels of putative cryoprotectants. Plasma levels of glucose and lactate, but not urea, were higher in cold-conditioned turtles than in controls, although the combined increase in these solutes was only 23 mmol L(-1). Cold conditioning attenuated cold-shock injury to brain cells, as assessed using a vital-dye assay, suggesting a link between protection of the nervous system and cold hardiness at the organismal level.