Carnitine dehydrogenase from Pseudomonas sp. YS-240 was purified to apparent homogeneity. Purification was achieved by ion-exchange chromatography, followed by ammonium sulfate fractionation, hydroxylapatite chromatography and gel filtration. This enzyme had the molecular weight of 110 kDa and consisted of two identical subunits. The isoelectric point was found to be 5.5. The optimum pH for enzymatic activity in the oxidation reaction was found to be 9.5. The enzyme was highly specific for L-carnitine and NAD(+). The Michaelis constants for L-carnitine and NAD(+) were 1.2 mM and 0.1mM, respectively.