The enzymatic system mainly responsible for the reduction of labile iron ions in mammalian cells is still unknown. Using isolated organelles of the rat liver, i.e. mitochondria, microsomes, nuclei and the cytosol, we here demonstrate that Fe(III), added as Fe(III)-ATP complex, is predominantly reduced by an NADH-dependent enzyme system associated with mitochondria (65% of the overall enzymatic Fe(III) reduction capacity within liver cells). Microsomes showed a significantly smaller Fe(III) reduction capacity, whereas the cytosol and nuclei hardly reduced Fe(III). Studying the mitochondrial iron reduction, this NADH-dependent process was not mediated by superoxide, ascorbic acid, or NADH itself, excluding low-molecular-weight reductants. No evidence was found for the involvement of complex I and III of the respiratory chain. Submitochondrial preparations revealed the highest specific activity reducing Fe(III) in the outer membrane fraction. In conclusion, an NADH-dependent mitochondrial enzyme system, most likely the NADH-cytochrome c reductase system, located at the outer membrane, should decisively contribute to the enzymatic reduction of labile iron within liver cells, especially under pathological conditions.