Polar fish, cold hardy plants, and overwintering insects produce antifreeze proteins (AFPs), which lower the freezing point of solutions noncolligatively and inhibit ice crystal growth. Fish AFPs have been shown to stabilize membranes and cells in vitro during hypothermic storage, probably by interacting with the plasma membrane, but the mechanism of this stabilization has not been clear. We show here that during chilling to nonfreezing temperatures the alpha-helical AFP type I from polar fish inhibits leakage across model membranes containing an unsaturated chloroplast galactolipid. The mechanism involves binding of the AFP to the bilayer, which increases the phase transition temperature of the membranes and alters the molecular packing of the acyl chains. We suggest that this change in acyl chain packing results in the reduced membrane permeability. The data suggest a hydrophobic interaction between the peptide and the bilayer. Further, we suggest that the expression of AFP type I in transgenic plants may be significant for thermal adaptation of chilling-sensitive plants.