The 5' region of the lac operon of Lactobacillus casei has been investigated. An open reading frame of 293 codons, designated lacT, was identified upstream of lacE. The gene product encoded by lacT is related to the family of transcriptional antiterminator proteins, which includes BglG from Escherichia coli, ArbG from Erwinia chrysanthemi, SacT, SacY, and LicT from Bacillus subtilis, and BglR from Lactococcus lactis. Amino acid sequence identities range from 35 to 24%, while similarities range from 56 to 47%. The transcriptional start site of the lac operon was identified upstream of lacT. The corresponding mRNA would contain in the 5' region a sequence with high similarity to the consensus RNA binding site of transcriptional antiterminators overlapping a sequence capable of folding into a structure that resembles a rho-independent terminator. LacT was shown to be active as an antiterminator in a B. subtilis test system using the sacB target sequence. lacT directly precedes lacEGF, the genes coding for enzyme IICB, phospho-beta-galactosidase, and enzyme IIA, and these genes are followed by a sequence that appears to encode a second rho-independent transcription terminator-like structure. Northern hybridizations with probes against lacT, lacE, and lacF revealed transcripts of similar sizes for the lac mRNAs of several L. casei strains. Since the length of the lac mRNA is just sufficient to contain lacTEGF, we conclude that the lac operon of L. casei does not contain the genes of the accessory tagatose-6-phosphate pathway as occurs in the lac operons of Lactococcus lactis, Streptococcus mutans, or Staphylococcus aureus.