The gram-positive, aerobic, moderately halophilic bacterium Halobacillus halophilus is challenged in its environment by frequently changing salt (NaCl) concentrations. Recently, H. halophilus was shown to be the first prokaryote that is dependent on Cl(-) for growth. In a search for the biological function of Cl(-) in this prokaryote, we identified different Cl(-)-dependent processes, which suggests a more general role for Cl(-) in the metabolism of H. halophilus. To analyze the effect of Cl(-) in more detail, we concentrated on one model system, the Cl(-)-dependent production of flagella, and aimed to identify the molecular basis for the Cl(-) dependence of flagellum production. Here, we report that synthesis of the major subunit of the flagellum, FliC, is dependent on the Cl(-) concentration of the medium, as determined by Western blot analyses. The gene encoding FliC was cloned and sequenced, and Northern blot as well as reverse transcriptase PCR analyses revealed that expression of fliC is Cl(-) dependent. FliC is the first protein of known function demonstrated to be synthesized in a Cl(-)-dependent manner in a prokaryote. Two-dimensional gel electrophoresis of cells grown under different conditions revealed five more Cl(-)-induced proteins; these were identified by N-terminal sequencing and database searches to be orthologs of proteins involved in stress response in Bacillus subtilis. The data indicate that Cl(-) is an important environmental signal in this moderate halophile and regulates protein synthesis and gene expression. Furthermore, the data may suggest that Cl(-) plays a role in the signal transduction involved in salt perception by this bacterium.