Biotin is a cofactor for carboxylases used in fatty acid synthesis, gluconeogenesis, and energy production by the citric acid cycle. Although lung has low levels of this vitamin overall, high concentrations were demonstrated histochemically in Clara cells of mouse, rat, hamster, and guinea pig using avidin conjugated to peroxidase. Lesser concentrations were found in type II cells of mouse, rat, and hamster but not guinea pig. By electron microscopy, biotin stores in mouse Clara cells were localized to mitochondria, while those in type II cells were present in both mitochondria and the cytoplasmic matrix. Biotin stores in type II cells are probably used mainly in fatty acid synthesis but also in gluconeogenesis and energy production. The reason for particularly high concentrations in the mitochondria of Clara cells is unknown.