In enteric bacteria, chromosomes are partitioned into domains that exhibit restricted supercoil movement. The most common domain barrier detected by gammadelta resolution assays is random with respect to sequence and occurs more frequently in cells growing rapidly in rich medium compared to cells in stationary phase. Transcription generates both positive and negative supercoiling movement. To address the question of whether transcription causes the appearance of new domain boundaries, a transcriptionally active MudI element was substituted for a MudJr-1 element that resides within the cobT gene of Salmonella typhimurium. Mu-specific transcription from the phage early promoter was placed under control of either the wild type (c(+)) or the temperature-sensitive (cts62) repressor. Using a resolution assay with res sites at six chromosomal locations, domain structure was normal in cells carrying the MudAr-1 prophage with a wild type Mu repressor. However, in cells with a MudAr-1 prophage harboring the cts62 repressor, a new domain barrier appeared in > 90% of the cells. Supercoil movement was restricted ahead of but not behind the transcription machinery. We conclude that the strong Mu early promoter induces the appearance of a domain barrier within the limits of a MudAr-1 prophage.