L(-)-Carnitine:NAD+ oxidoreductase, EC 1.1.1.108, from Agrobacterium sp. catalyzes the oxidation of L(-)-carnitine to 3-dehydrocarnitine as initial step of L(-)-carnitine degradation. The enzyme was purified 76-fold by four chromatographic steps. A high substrate specificity for L(-)-carnitine and NAD+ was observed. The molecular mass of the native enzyme is 114 kDa and it consists of two identical subunits as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point was found to be 5.2-5.4. The optimum temperature is 45 degrees C and the optimum pH for the oxidation and the reduction reaction are 9.5 and 5.5-6.5, respectively. Kinetic parameters and amino-terminal sequence were determined. The oxidation reaction is inhibited by D(+)-carnitine, trimethylamine, several metal ions and cetyltrimethylammoniumbromide (CTAB).