The topology of 4-pyridoxic acid dehydrogenase in the Escherichia coli cell membrane was examined with transformed E. coli cells overexpressing the enzyme from Mesorhizobium loti. The recombinant enzymes with a His(6)-tag either in the N-terminal region or at the C-terminus were localized on the E. coli cell membrane like the wild-type enzyme without a His(6)-tag. The His(6)-tags were labelled with Ni-NTA AP conjugate only when the E. coli protoplast cells were broken. The membrane-bound enzyme in the intact protoplast cells was not digested by trypsin, although the one in the gently broken protoplast cells was almost totally digested. Thus, 4-pyridoxic acid dehydrogenase was an integral monotopic protein, protruding into a cytoplasm side from the bacterial membrane. The deletion or mutation of a deduced transmembrane segment in 4-pyridoxic acid dehydrogenase made it an inclusion body, and the enzyme protein was not found in the E. coli cell membrane. Thus, it was suggested that the intact deduced transmembrane segment was necessary for 4-pyridoxic acid dehydrogenase to be localized on the bacterial cell membrane.