Freezing conditions naturally induce chronic photoinhibition (ie light-dependent decrease in efficiency of photosystem II) in over-wintering leaves of eucalypts and other evergreen species. We examined relationships between spatial patterns in freezing, concentrations of photosynthetic pigments, and photosynthetic characteristics. Patterns of freezing in intact, attached leaves of Eucalyptus pauciflora were studied with infra-red thermal imaging. After ice nucleation, temperatures increased to values near the freezing point over the whole leaf. However, local variation in leaf shape, thickness and water content influenced the duration of elevated temperatures, causing spatial gradients in leaf temperatures during freezing of as much as 4° C. Temperatures were lower (and hence dehydration greater) toward the leaf tip and margins. Spatial patterns in pigmentation in leaves that had been exposed to naturally occurring frosts were determined by conventional extraction techniques combined with high resolution hyperspectral imaging of reflectance from intact leaves. These measurements showed that concentrations of chlorophyll per leaf area were lower while concentrations of zeaxanthin plus antheraxanthin per chlorophyll were higher toward the leaf tip and margins. These changes in pigment composition were highly correlated with greater sustained reduction in Fv/Fm, which in turn was strongly correlated with a unique emission band in the 77K fluorescence spectra signifying the reversible formation of a recently discovered novel photoprotective complex (Gilmore and Ball 2000 PNAS 97: 11098-11101). Thus, altered patterns in pigmentation and thylakoid function were consistent with patterns in freezing temperatures. These data provide explicit spatial linkage between environmental stress and physiological responses to it.