We used a combined computer and biochemical approach to characterize human pyridoxine 5'-phosphate oxidase (PNPO). The human PNPO gene is composed of seven exons and six introns, and spans approximately 8 kb. All exon/intron junctions contain the gt/ag consensus splicing site. The absence of TATA-like sequences, the presence of Sp1-binding sites and more importantly, the presence of CpG islands in the regulatory region of the PNPO gene are characteristic features of housekeeping genes. Northern blot analyses showed two species of poly(A)(+) RNA of approximately 2.4 and approximately 3.4 kb at identical intensity, whereas Western blot analysis showed that no protein isoform exists in any of the tissues examined. PCR-based analysis led to the idea that two messages are transcribed from a single copy gene, and that the size difference is due to differential usage of the polyadenylation signal. The major sites of PNPO expression are liver, skeletal muscle and kidneys while a very weak signal was detected in lung. The mRNA master dot-blot for multiple human tissues provided a complete map of the tissue distribution not only for PNPO but also for pyridoxal kinase and pyridoxal phosphatase. The data indicate that mRNA expression of all three enzymes essential for vitamin B(6) metabolism is ubiquitous but is highly regulated at the level of transcription in a tissue-specific manner. In addition, human brain PNPO cDNA was expressed in Escherichia coli, and the roles of both the N- and C-terminal regions were studied by creating sequential truncation mutants. Our results showed that deletion of the N-terminal 56 residues affects neither the binding of coenzyme nor catalytic activity.