Vitamin B6 is an essential cofactor for more than 100 enzymatic reactions. Mammalian cells are unable to synthesize vitamin B6 de novo, whereas bacteria, plants, fungi, and as shown here Plasmodium falciparum possess a functional vitamin B6 synthesis pathway. P. falciparum expresses the proteins Pdx1 and Pdx2, corresponding to the yeast enzymes Snz1-p and Sno1-p, which are essential for the vitamin B6 biosynthesis. An involvement of PfPdx1 and PfPdx2 in the de novo synthesis of vitamin B6 was shown by complementation of pyridoxine auxotroph yeast cells. Both plasmodial proteins act together in the glutaminase activity with a specific activity of 209 nmol min(-1) mg(-1) and a K(m) value for glutamine of 1.3 mm. Incubation of the parasites with methylene blue revealed by Northern blot analysis an elevated transcriptional level of pdx1 and pdx2, suggesting a participation of these proteins in the defenses against singlet oxygen. To be an active cofactor, vitamin B6 has to be phosphorylated by the pyridoxine kinase (PdxK). The recombinant plasmodial PdxK revealed K(m) values for the B6 vitamers pyridoxine and pyridoxal and for ATP of 212, 70, and 82 microM, respectively. All three enzymes expose a stage-specific transcription pattern within the trophozoite stage that guarantees the concurrent expression of Pdx1, Pdx2, and PdxK for the indispensable provision of vitamin B6. The occurrence of the vitamin B6 de novo synthesis pathway displays a potential new drug target, which can be exploited for the development of new chemotherapeutics against the human malaria parasite P. falciparum.