The trp genes of Brevibacterium lactofermentum lie within a 7.72-kb HapII-BamHI fragment whose sequence has been determined (Matsui et al., 1986). The 5'- and the 3'-flanking regions of this gene cluster were subcloned as a 1.8-kb PstI-PstI and a 0.6-kb XhoI-BamHI fragment, respectively. The 5'-flanking region encodes two open reading frames (ORFs); one corresponds to trpL, while the other corresponds to the N-terminal half of the trpE gene. Within the 17 amino acid residues of the predicted leader peptide encoded by trpL are found three contiguous tryptophan residues. By subcloning parts of the 1.8-kb PstI-PstI fragment into promoter probe vectors, a promoter situated 32 bp upstream from the presumptive trpL gene was identified. The -35 and the -10 region of this promoter, TACACA and AATAAT, respectively, are very similar to the Escherichia coli trp promoter. The trp promoter of B. lactofermentum functions in E. coli. A 14-bp imperfect palindrome that overlaps the -10 region apparently functions in B. lactofermentum but not in E. coli as a trp operator. Upstream of trpE, attenuator-like sequences are found that resemble the corresponding E. coli sequences. The 0.6-kb XhoI-BamHI 3'-flanking fragment contains one ORF that encodes the C-terminal part of trpA. Downstream from trpA lies a Rho-independent terminator that resembles E. coli sequences that are situated downstream from the E. coli trp operon. Thus the trp control regions of the Gram-positive B. lactofermentum are more closely related in structure to the corresponding regions of the Gram-negative E. coli than to those of the Gram-positive Bacillus subtilis.