Technical Abstract: It has previously been reported that moderately high dietary manganese (Mn) in combination with marginal magnesium (Mg) resulted in ultrastructural damage to heart mitochondria. Manganese may replace Mg in biological functions, including the role of enzyme cofactor. Manganese may accumulate and substitute for Mg during the condition of Mg-deficiency. The objective of the current study was to determine whether high dietary Mn alters heart muscle respiration and Mg-enzyme activity as well as whole body Mn retention under marginal Mg. An additional objective was to determine whether high Mn results in increased oxidative stress. In experiment 1: forty-eight rats were fed a 2 X 3 factorial arrangement of Mn (10, 100, or 1000 mg/kg) and Mg (200 or 500 mg/kg). In experiment 2: thirty-two rats were fed one of four diets in a 2 X 2 factorial arrangement of Mn (10 or 250 mg/kg) and Mg (200 or 500 mg/kg). In experiment 3: thirty-two rats were fed one of four diets in a 2 X 2 factorial arrangement of Mn (10 or 650 mg/kg) and Mg (200 or 500 mg/kg). In experiment 2, high Mn and marginal Mg reduced (P < 0.05) oxygen consumption of left ventricle muscle. Marginal Mg, but not Mn, reduced (P < 0.05) activity of sarcoplasmic reticulum calcium-ATPase enzyme. Dietary Mg had no affect on (54)Mn kinetics, but high dietary Mn decreased (P < 0.01) absorption, retention, and rate of excretion of (54)Mn. Neither cellular stress, measured by Comet assay, nor antioxidant enzyme activities were increased by high Mn. A strong interaction (P < 0.001) between increasing Mn and adequate Mg on hematology was observed. These results confirm previous research in swine that high Mn alters myocardial integrity as well as function, but not as a result of altered calcium transport or oxidative stress.