The genes coding for threonyl-tRNA synthetase (thrS), translation initiation factor 3 (infC) and ribosomal protein L20 (rplT) are clustered in the Escherichia coli genome. Previous studies had suggested the possibility that the expression of these genes is coupled. The transcriptional events in this operon have now been examined by S1 nuclease mapping and promoter fusion studies. The results indicate that infC-containing mRNAs are initiated from three separate promoters. Two of these are located in the protein-coding region of thrS and one, P12, is the major promoter at all growth rates tested. In addition, there is co-transcription of thrS and infC from the thrS promoter (PT). A single promoter for thrS has been mapped approx. 170 nucleotides upstream from its translation initiation site. Another promoter has been located within the infC-coding region. It is separated from the next downstream gene, rplT, by a transcription end point. However, termination at this region is only 50-70% efficient and transcripts starting at this promoter can read through into rplT. These findings demonstrate that the pattern of transcription in this operon is highly complex and the mRNA levels for each of the genes is determined by a variety of factors, including multiple promoters, co-transcription and readthrough of transcription termination signals.