Objectives: Human novel oxidoreductase 1 (NDOR1) is a diflavin reductase closely related to cytochrome P450 reductase (POR) and nitric oxide synthase (NOS), which are involved in the metabolism of antitumour agents. A variant cDNA sequence of NDOR1, NDOR1 p.518-519ins9 or NDOR1_v1, has been deposited in GenBank (accession no. AK026089 and AY077845) that encodes an additional nine amino acids, which led us to investigate NDOR1 polymorphism. Methods and results: We analysed genomic DNA from 200 Caucasian and 49 Japanese individuals by PCR–restriction fragment length polymorphism analysis. The nine amino acid residue sequence was found to be present in all individuals, encoded by a 27 nt intronic nucleotide sequence at an intron/exon junction, suggesting that this variant did not represent a common genetic polymorphism, but may have arisen from an alternative mRNA splicing event. However, further analysis of NDOR1 revealed a polymorphic c.1564G>A transition (NDOR1*1), detected in 24/200 Caucasian and 1/49 Japanese individuals, producing a valine to isoleucine substitution at codon 522 in the NADPH binding region. Expression of the flavin adenine dinucleotide/reduced nicotinamide phosphate dehydrogenase (NADPH) domain in Escherichia coli showed a significant 74% reduction in potassium ferricyanide reductase activity, but no effect on NADPH binding. NDOR1_v1 showed a 10-fold decrease in affinity for NADPH, and a 90% reduction in ferricyanide reductase activity. Conclusions: We have discovered a polymorphic variant of NDOR1, NDOR1*1, that produces a functionally impaired enzyme. This will help define the structure and function of NDOR1 and its relationship to cancer and other diseases.