Weanling mice were fed a riboflavin-deficient diet or the same diet with added galactoflavin. Both diets produced changes in hepatic mitochondrial morphology, the most striking of which was the development of giant mitochondria. The livers from these animals were fractionated, and the nuclear and mitochondrial fractions were examined by electron microscopy. The nuclear fraction contained giant mitochondria; the mitochondrial fraction contained the remaining normal to moderately enlarged mitochondria. Oxidative studies were carried out on the mitochondrial fractions. It was found that both experimental diets resulted in a marked reduction in fatty acid oxidation by the mitochondria. In addition, the mitochondria of mice with advanced riboflavin deficiency (induced simply by a riboflavin-free diet) showed severely decreased state 3 (ADP-stimulated) respiration and depressed respiratory control ratios, but normal ADP/O ratios. In contrast, mitochondrial performance (aside from fatty acid oxidation) in galactoflavin-supplemented, riboflavin-deficient mice was related to the gross appearance, i.e., color, of the liver from which these organelles were derived. In mice fed this diet, the livers were either red or yellow. Mitochondria from yellow livers showed normal oxidative phosphorylation. Mitochondria from red livers showed a serious reduction in state 3 oxidation. This study demonstrates that in the mouse, riboflavin deficiency, however produced, not only results in altered mitochondrial morphology but also results in significantly impaired mitochondrial function.