A physical barrier exists in Vitis vinifera L. (wine-quality grapevines) between the canes that freeze at subzero temperatures and the buds that supercool, thereby avoiding ice formation in these delicate organs. Our objective was to characterize the ice nucleation barrier by observing grape buds at different stages of acclimation, by treating them with pectinase, and by comparing them to buds of the more hardy Vitis riparia L. and to non-supercooling buds of Populus nigra L. "Italica." Differential thermal analysis (DTA) indicated that as V. vinifera andV. riparia acclimated in the autumn, the ice nucleation point of the buds declined, then increased as the grapevines de-acclimated in the spring. Laser scanning confocal microscopy was used to detect the penetration of fluorescent rhodamine green dyes from the cane into the bud as a measure of apoplastic permeability. These pore size exclusion tests indicated that the bud axes were impermeable to a 3000 MW dextran-conjugated dye when the ice nucleation temperature was below -20°C, but permeable above -20°C. This lower porosity presumably restricts the penetration of ice into the bud from the cane. In contrast to the two Vitis species, the permeability of Populus buds did not limit ice propagation, and the water in buds froze between -5 and -10°C according to DTA. Pectinase and phosphate treatments of grape buds to remove pectin increased apoplastic permeability, increased the ice nucleation temperature, and prevented supercooling according to DTA. Light microscopy indicated that suberin coated the scales of the grape buds, probably preventing ice nucleation from the environment, but was not present in the bud axis region. We concluded that a permeability barrier, possibly containing pectin, in the axis of grape buds limits the propagation of ice into the buds and enables supercooling to occur.Key words: supercooling, freezing, acclimation, winter hardiness, pectin, confocal microscopy.