L-Carnitine dehydrogenase (CDH) was partially purified from Pseudomonas putida IAM12014 for the stereospecific reduction of 3-dehydrocarnitine to L-carnitine. CDH and glucose dehydrogenase (GDH) were coimmobilized in a nanofiltration membrane bioreactor (NFMBR) for the continuous production of L-carnitine from 3-dehydrocarnitine with NADH regeneration. In the NFMBR, NAD was partially immobilized through rejection by the nanofiltration membrane and effectively regenerated by the conjugation reaction of CDH and GDH. Since 3-dehydrocarnitine was unstable at neutral pH, it was maintained under acidic conditions (pH 0.7) and supplied to the NFMBR separately from the other substrates, glucose and coenzyme NAD. As 50 mM 3-dehydrocarnitine in HCl solution, 0.05 mM NAD, and 100 mM glucose in 0.5 M Tris buffer (pH 8) were continuously supplied to the NFMBR with immobilized CDH (200 U/ml) and GDH (200 U/ml) at the retention time of 80 min and temperature of 25 degrees C, the maximum conversion, reactor productivity, and NAD regeneration number were 78%, 113 g/l/d, and 780, respectively. The half-life of the NFMBR was longer than 500 h.