This investigation represents a first attempt to gain a quantitative estimate of the effects of. the anions sulfate, citrate, acetate, chloride and thiocyanate on the thermodynamic stability (AG ') of a model globular protein in ice at -15 'C. The method, based on guanidiniurn chloride denaturation of the azurin mutant CI 12S from Pseudonionas aeruginosa, distinguishes between the effects of cooling to subfreezing temperatures from those induced specifically by the formation of a solid ice phase. The results confirm that, both in liquid and frozen states, kosmotropes (sulfate, citrate and acetate) increase significantly protein stability, relative to chloride, whereas the chaotrope thiocyanate decreases it. Throughout, their stabilizing efficacy was found to rank according to the Hofmeister series, sulfate > citrate > acetate > chloride > thiocyanate, although the magnitude of A(AG') exhibited a distinct sensitivity among the anions to low temperature and to ice formation. In the liquid state, lowering the temperature from +20 to - 15 'C weakens considerably the stabilizing efficacy of the organic anions citrate and acetate. Among the anions sulfate stands out as the only strong stabilizer at subfreezing temperatures while SCN- becomes an even stronger denaturant. Freezing of the solution in the presence the "neutral" salt NaCl destabilizes the protein, AG' progressively decreasing up to 3-4 kcal/mol as the fraction of liquid water in equilibrium with ice (VL) is reduced to less than 1%. Kosmotropes do attenuate the decrease in protein stability in ice although in the case of citrate and acetate, their efficacy diminishes sharply as the liquid fraction shrinks to below 2.7%. On the contrary, sulfate is remarkable for it maintains constantly high the stability of azurin in liquid and frozen solutions, down to the smallest VL (0.5%) examined. Throughout, the reduction in AG' caused by the solidification of water correlates with the decrease in the denaturant ni value, an indirect indication that protein-ice interactions generally lead to partial unfolding of the native state. It is proposed that binding of the kosmotropes to the ice interface may inhibit protein adsorption to the solid phase and thereby counter the ice perturbation.