Two protein factors from hardy leaves were highly active in preventing the inactivation of photophosphorylation of washed thylakoid membranes which takes place during freezing of thylakoids in the absence of protective compounds. Non-hardy leaves did not yield protective protein material. On a unit weight basis, protection by the protein factors was 10 to 100 times better than protection by compounds of low molecular weight such as sucrose, glycerol or dimethylsulfoxide. Very low amounts of the protein factors, which alone were scarcely protective, considerably reduced the concentration of sucrose required for the complete protection of thylakoids during freezing. The protein factors were heat-stable and had a molecular weight between 10 000 and 20 000 daltons. It is assumed that they contribute to and are in part responsible for the frost tolerance of hardy plant material.