The relative biological value (RBV) of iron in egg yolk was compared with that of iron in ferrous sulfate in prophylactic assays with weanling rats. Egg yolk cooked by one of three different methods was used in each experiment: (1) yolk of eggs boiled in the shell, lyophilized, and mixed with other dry diet ingredients; (2) pasteurized yolk and other diet components steamed with 8 liters of water per kilogram of diet; and (3) pasteurized yolk and other diet ingredients baked with 0.5 liters of water per kilogram of diet. Diets containing three levels of egg yolk and four levels of ferrous sulfate were fed to different groups of rats for 1, 2, and 3 weeks. Response of dietary iron was calculated as the regression of hemoglobin iron gain on iron intake and the nutritional quality of egg iron relative to that of ferrous sulfate iron was evaluated by slope-ratio analysis. RBV was 61, 64, and 90%, respectively, for iron of egg yolk cooked by the three methods. The presence of ascorbic acid during heat treatment of the diets significantly increased RBV of yolk iron from 64 to 92% in experiment 2, and increased it to a value equivalent to that of ferrous sulfate in experiment 3.