L-Lysine-alpha-ketoglutarate reductase (N5-(1,3-dicarboxypropyl)-L-lysine: NADP+ oxidoreductase (L-lysine-forming, EC 1.5.1.8) was purified from rat liver mitochondria to a homogeneous state judged by SDS polyacrylamide gel electrophoresis, and its molecular weight was estimated as 52000. On Sepharose 4B filtration it has a molecular weight of 230 000 and it is suggested that the active enzyme is a tetramer of subunits of similar size. The purified enzyme was clearly separated from saccharopine dehydrogenase (N5-(1,3-dicarboxypropyl)-L-lysine:NAD+ oxidoreductase (L-glutamate-forming, EC 1.5.1.9). The reactions of purified L-lysine-alpha-ketoglutarate reductase favored the forward reaction (saccharopine formation) and the rate of the reverse reaction (lysine formation) was only 3--5% that of the forward reaction. The forward reaction was specific for L-lysine, alpha-ketoglutarate and NADPH and followed Michaelis-Menten kinetics, whereas the dose vs. response curve of the reverse reaction was sigmoidal with saccharopine. Among the amino acids examined, ornithine, leucine and tryptophan inhibited the forward reaction competitively. These results are different from earlier reports on human and yeast enzymes. The fact that rats fed on lysine-deficient diet do not lose weight much is discussed in relation to the properties of this enzyme.