The effects of copper deficiency on the proteins of rat erythrocyte membranes were assessed by electrophoretic analysis. For 42 d, rats were fed diets containing less than 1 ppm Cu with 35 or 250 ppm Fe or 5 ppm Cu with 35 or 250 ppm Fe. Electrophoresis of erythrocyte membrane proteins indicated a significant increase in the amount of a 170,000-dalton protein in rats fed copper-deficient diets. High dietary iron reduced the amount of the 170,000-dalton protein in erythrocyte membranes from rats in both the copper-deficient and copper-adequate groups. However, feeding high dietary iron to copper-deficient rats did not reduce the amount of the protein to the level found in rats fed the copper-adequate diet containing 35 ppm Fe. Triton X-100 extraction of erythrocyte membranes demonstrated that the 170,000-dalton protein was associated with the membrane cytoskeleton. Thus, copper deficiency possibly alters the cell's mechanical properties and consequently decreases erythrocyte survivability by modifying the membrane cytoskeleton.