Vibrio cholerae is known to secrete a large number of proteins into the extracellular milieu, including the important virulence factor cholera toxin (CT). However, one of the most abundant proteins found in V. cholerae supernatants is the zinc-metalloprotease HA/protease (HAP). Whereas efficient protein secretion in Escherichia coli requires ATP hydrolysis and the proton motive force (pmf), little is known about the energy requirements for protein secretion in V. cholerae. To analyze some of the energy requirements for protein secretion in V. cholerae, HAP accumulation in culture supernatants following growth in the presence of various ionophores was assayed. Extracellular production of HAP was strongly reduced in the presence of monensin, an artificial Na(+)/H(+) antiporter that collapses the DeltapNa(+) across the membrane without affecting Deltapsi, whereas the protonophore CCCP had no significant effect on the extracellular accumulation of HAP. In contrast, extracellular protease production in Pseudomonas aeruginosa was affected by CCCP, but not monensin. Furthermore, extracellular protease production of V. cholerae, but not P. aeruginosa, was increased in increasing amounts of NaCl in the culture medium. Together these results indicate that the V. cholerae HAP requires an intact sodium motive force (smf) for its efficient translocation across the membranes, whereas extracellular protease production by P. aeruginosa requires only pmf. As the entry of some bacteriophage genomes has been reported to require pmf, the effects of ionophores on the efficiency of tranduction of V. cholerae by the CTXPhi phage were analyzed. CTXPhi transduction was strongly affected by CCCP, but not monensin, suggesting that phage entry requires pmf but not smf. Understanding the energy requirements for these potentially important virulence aspects of pathogens might lead to novel intervention strategies.