Abstract 1. 1. Phosphofructokinase from frog skeletal muscle has been isolated and purified to homogeneity. 2. 2. The sensitivity of this poikilothermic enzyme to temperature inactivation has been investigated. 3. 3. The inactivation of the enzyme as a function of pH is markedly enhanced at pH values less than 7.0 and at low temperatures. ATP also enhances inactivation under these conditions, while fructose-1,6- bisphosphate dramatically stabilizes the enzyme. 4. 4. The regulatory kinetic behavior (ATP inhibition and sigmoidal kinetics with fructose-6-phosphate) at pH 6.73 are demonstrated to be temperature sensitive. 5. 5. The cold lability of this enzyme is discussed in relation to the slowing of the metabolic rate of poikilotherms at decreased temperatures.