Recently, we reported the presence of ice nucleating activity, apparently proteinaceous, in the plasma of a freeze-tolerant frog, Rana sylvatica, collected in autumn and spring. Although this protein has not been purified, its ice nucleating behavior can act as an internal reference for tests that attempt to modify its ability to nucleate ice formation. If the addition of a chemical reagent alters the temperature of ice crystallization compared with the control, it can be assumed that protein modification may have occurred. The ice nucleating protein in R. sylvatica showed resistance to proteolysis with four different proteases although there was a significant reduction in the temperatures of nucleation with these treatments (ANOVA P less than 0.001). However, ice nucleating activity was lost when plasma was treated with the addition of urea or N-bromosuccinimide. Modification of protein sulphydryl groups with iodoacetamide did not affect the crystallization temperature (Tc) but treatment with iodoacetic acid resulted in a significant increase in Tc of plasma. An abrupt loss of ice nucleating ability was observed in plasma samples after heating above 87 degrees C. Anomalous potentiation of ice nucleating activity occurred when the plasma was heated to and held at temperatures between 67-75 degrees C.