One of the strategies used by organisms to adapt to life under conditions of short energy supply is to use the by-product pyrophosphate to support cation gradients in membranes. Transport reactions are catalyzed by membrane-integral pyrophosphatases (PPases), which are classified into two homologous subfamilies: H
+
-transporting (found in prokaryotes, protists, and plants) and Na
+
-transporting (found in prokaryotes). Transport activities have been believed to require specific machinery for each ion, in accordance with the prevailing paradigm in membrane transport. However, experiments using a fluorescent pH probe and
22
Na
+
measurements in the current study revealed that five bacterial PPases expressed in
Escherichia coli
have the ability to simultaneously translocate H
+
and Na
+
into inverted membrane vesicles under physiological conditions. Consistent with data from phylogenetic analyses, our results support the existence of a third, dual-specificity bacterial Na
+
,H
+
-PPase subfamily, which apparently evolved from Na
+
-PPases. Interestingly, genes for Na
+
,H
+
-PPase have been found in the major microbes colonizing the human gastrointestinal tract. The Na
+
,H
+
-PPases require Na
+
for hydrolytic and transport activities and are further activated by K
+
. Based on ionophore effects, we conclude that the Na
+
and H
+
transport reactions are electrogenic and do not result from secondary antiport effects. Sequence comparisons further disclosed four Na
+
,H
+
-PPase signature residues located outside the ion conductance channel identified earlier in PPases using X-ray crystallography. Our results collectively support the emerging paradigm that both Na
+
and H
+
can be transported via the same mechanism, with switching between Na
+
and H
+
specificities requiring only subtle changes in the transporter structure.