We isolated a miniTn10(Cmr) insertion mutant lacking pyridoxine (PN) kinase and cloned the structural gene, designated pdxK, by complementation. P1 transduction and PCR mapping and DNA sequence analysis showed that pdxK was adjacent to the crr sugar transport gene (53.95 min). Growth properties of pdxK::miniTn10 mutants supported the hypotheses that PN kinase, which also phosphorylates pyridoxal (PL) and pyridoxamine (PM) in vitro, functions solely in the B6-vitamer salvage pathway and that E. coli contains an additional PL kinase. The amino acid sequence of PdxK has signature motifs of the PfkB superfamily of carbohydrate kinases, which includes phosphofructokinases and ribokinases, and suggests that three unidentified ORFs of Salmonella typhimurium, Haemophilus influenzae, and Saccharomyces cerevisiae correspond to PN/PL/PM kinases.