A transcriptional analysis was undertaken for the toxigenic element of Clostridium difficile in five strains which differ greatly in toxigenicity. The toxigenic element has recently been described in C. difficile strain VPI 10463 and consists of three small open reading frames in addition to the toxin A and B genes. A large, polycistronic transcript (17.5 kb) was detected, in addition to processing intermediates, and individual transcripts for toxin A, toxin B, and two of the three small open reading frames. A transcription initiation site and a promoter for the toxigenic element were identified, as well as major extension products upstream of the toxin A and B genes. These data support models in which the toxin A and B genes are cotranscribed along with the open reading frames, in addition to being transcribed individually. Transcriptional analyses, using probes for the transcripts for toxin A and toxin B, revealed quantitative differences among strains which correlated with quantitative differences in toxin production among these strains. However, DNA sequencing of intergenic regions in these strains showed remarkable DNA sequence conservation in these intergenic regions.