The gas vesicle formation in Haloferax mediterranei occurs in the stationary growth phase and involves the 14 genes mc-gvpACNO and mc-gvpDEFGHIJKLM. The appearance of the two regulatory proteins GvpD and GvpE, and also of GvpF, was investigated during the growth of H. mediterranei. GvpD was only found during the stationary growth phase, GvpE was present from the late exponential to stationary growth phase, and GvpF was present only during the exponential growth, although the three genes were co-transcribed. The impact of GvpD and GvpE on the activity of the promoter of the mc-gvpACNO gene cluster encoding the gas vesicle structural proteins was analysed in H. volcanii transformants containing the mc-gvpA gene or a fusion of the mcA promoter with the bgaH reading frame encoding a halobacterial beta-galactosidase as reporter. The experiments proved that GvpE is a transcriptional activator, whereas GvpD is involved in the repression. Protein-protein affinity chromatography was used to search for putative binding partners of GvpD and GvpE. Both proteins were synthesized in Escherichia coli as his-tagged proteins, isolated under denaturing conditions and refolded by dialysis against buffers containing decreasing urea and increasing KCl concentrations up to 2.5 M. The Ni-NTA matrix tagged with GvpD-his or GvpE-his was incubated with soluble proteins of gas vesicle producing H. mediterranei cells. A 21 kDa protein was purified using the matrix tagged with GvpD-his which proved to be GvpE by Western analysis. Vice versa, GvpD was purified using the GvpE-his-Ni-NTA matrix. These results strongly suggested that GvpD and GvpE were able to interact and might constitute a regulatory system.