Conjugative transfer of the Lactococcus lactis plasmid pRS01 requires splicing of a group II intron, Ll.ltrB, for accurate translation of the mRNA for the exon gene ltrB. The protein product of ltrB is a conjugative relaxase, essential for pRS01 transfer. Using a molecular technique for the identification of transcription initiation sites in bacteria, a functional promoter within Ll.ltrB was identified upstream from the gene for the intron-encoded protein (IEP) LtrA. LtrA Is required for efficient splicing of Ll.ltrB in vivo. Mutation of the ltrA promoter dramatically reduced the steady-state level of ltrA mRNA, LtrA, intron splicing and conjugation in L. lactis. These effects could be relieved by expression in trans of the ltrA gene cloned under the control of an inducible promoter. These results suggest that the ltrA mRNAs are translated inefficiently. We hypothesize that this bacterial intron, in contrast to previously studied group II introns in eukaryotes, requires a promoter within the intron to regulate ltrA expression and to produce an adequate level of the protein in the cell far efficient splicing.