Expression of the Escherichia coli proU operon, which encodes an efficient uptake system for the osmoprotectant glycine betaine, is strongly increased in cells grown at high osmolarity. We isolated 182 independent spontaneous mutants with elevated expression of the chromosomal phi(proV-lacZ) (Hyb2) fusion at low osmolarity. Genetic analysis demonstrated that eight of these mutant strains carried mutations closely linked to the fusion, whereas all others carried mutations that appeared to be in osmZ. All of the mutations resulted in increased but still osmoregulated expression of the phi(proV-lacZ)(Hyb2) fusion. The proU-linked mutants carried an identical point mutation (proU603) which changes the -35 sequence of the proU promoter from TTGCCT to TTGACT and thereby increases the homology of the -35 region to the consensus sequence (TTGACA) of E. coli promoters. We also selected for mutants with decreased expression of the plasmid pOS7-encoded phi(proV-lacZ)(Hyb2) fusion and isolated a plasmid with an IS1 insertion (proU607) between the proU -10 and -35 regions. This insertion creates a hybrid promoter and drastically reduces expression of the fusion but does not abolish its osmotic regulation. Deletion analysis of chromosomal sequences 5' to the proU promoter revealed that sequences located approximately 200 bp upstream of the -35 region were required for high-level expression. Removal of these sequences resulted in a 10-fold decline of phi(proV-lacZ)(Hyb2) expression. Osmotic regulation was retained in deletion constructs carrying just 19 bp of chromosomal DNA 5' of the promoter, showing that no sequences further upstream are required for the proper osmoregulation of proU transcription. Experiments with himA and fis mutant strains indicated that the IHF and FIS proteins are not required for the normal osmoregulation of proU expression.