Expression of the proU operon of Escherichia coli is directly proportional to the osmolarity of the growth medium. The basal level of proU transcription is very low, but a large increase is triggered by a sudden rise in the external osmolarity. This increased expression is maintained for as long as the osmotic stimulus persists. We have capitalized upon these regulatory features of the proU operon and have constructed a series of expression vectors (pOSEX) permitting osmotically controlled expression of heterologous genes governed by regulatory signals of proU. The pOSEX vectors carry the proU promoter, an upstream region required for high-level expression, and part of the first structural gene (proV), which acts as a silencer and is necessary to maintain low-level expression in low osmolarity media. An extended multiple cloning site (MCS) positioned at the 3' end of proV' permits the cloning of heterologous genes into the pOSEX plasmids, and efficient transcription terminators derived from the rrnB operon prevent deleterious read-through transcription into the vector portion. The properties of the pOSEX expression vectors were tested by positioning a promoterless lacZ (encoding beta-galactosidase) gene from E. coli and the gcdA (encoding carboxytransferase) gene from the Gram+ bacterium Acidaminococcus fermentans under the control of the proU regulatory region. Efficient, osmo-regulated and finely tuned expression of both lacZ and gcdA was achieved, and the amount of beta-galactosidase and carboxytransferase synthesized were simply controlled by adjusting the osmolarity of the growth medium with various concentrations of NaCl.