Differential thermal analysis, differential scanning calorimetry, pulsed nuclear magnetic resonance spectroscopy, and low temperature microscopy are utilized to investigate low temperature freezing points or exotherms which occur near -40 C in the xylem of cold-acclimated shagbark hickory (Carya ovata L.). Experiments using these methods demonstrate that the low temperature exotherm results from the freezing of cellular water in a manner predicted for supercooled dilute aqueous solutions. Heat release on freezing, nuclear magnetic resonance relaxation times, and freezing and thawing curves for hickory twigs all point to a supercooled fraction in the xylem at subfreezing temperatures. Calorimetric and low temperature microscopic analyses indicate that freezing occurs intracellularly in the xylem ray parenchyma. The supercooled fraction is found to be extremely stable, even at temperatures only slightly above the homogeneous nucleation temperature for water (-38 C). Xylem water is also observed to be resistant to dehydration when exposed to 80% relative humidity at 20 C. D(2)O exchange experiments find that only a weak kinetic barrier to water transport exists in the xylem rays of shagbark hickory.