The Escherichia coli Tat system serves to export folded proteins harbouring an N-terminal twin-arginine signal peptide across the cytoplasmic membrane. In this report we have studied the functions of conserved residues within the structurally related TatA and TatB proteins. Our results demonstrate that there are two regions within each protein of high sequence conservation that are critical for efficient Tat translocase function. The first region is the interdomain hinge between the transmembrane and the amphipathic alpha-helices of TatA and TatB proteins. The second region is within the amphipathic helices of TatA and TatB. In particular an invariant phenylalanine residue within TatA proteins is essential for activity, whereas a string of glutamic acid residues on the same face of the amphipathic helix of TatB is important for function.