Many Gram-negative bacteria naturally produce membrane vesicles (MVs) to the extracellular milieu. The Pseudomonas quinolone signal (PQS), a quorum-sensing signal of Pseudomonas aeruginosa, is a positive regulator of MV production. In this study, we investigated its effects on MV production in other Gram-negative and -positive bacterial species. The addition of PQS to an Escherichia coli K12 culture resulted in increased MV production and enlarged MVs. An excessive amount of MgCl(2) repressed E. coli MV production either with or without PQS, suggesting that an anionic repulsion of cellular surfaces increases MV production. PQS was found in the cellular membrane and MVs in E. coli. The enhancement of MV production by PQS occurred in other Gram-negative bacteria, including Burkholderia and Pseudomonas species. Moreover, PQS induced MV production in a Gram-positive bacterium, Bacillus subtilis 168, which does not normally produce MV under laboratory conditions. An excessive amount of MgCl(2) did not repress B. subtilis MV production in the presence of PQS, suggesting the production mechanism to be different from that in Gram-negative bacteria. Together, these results indicated that PQS enhances MV production in Gram-negative bacteria and induces it in Gram-positive bacteria.