In vitro transcription experiments were carried out with recombinant plasmids containing the promoters of the rrnB gene of Escherichia coli, and with deletion mutants lacking various lengths of the AT-rich sequence upstream from the P1 promoter of that gene. The main conclusions are as follows: The in vitro transcriptional activity of the P1 and P2 promoters of the rrnB gene are an order of magnitude higher on closed-circular (supercoiled) templates than on linear DNA; the strong P1 and P2 promoters are heparin-sensitive on linear templates, and on circular DNA only P2 is heparin-resistant; removal of the upstream AT-rich region did not decrease the apparent in vitro strength of the P1 promoter under standard conditions (50 mM KCl, high RNA polymerase/DNA ratio); at higher salt concentrations, or with a lower RNA polymerase/DNA ratio, the deletion mutants displayed much lower in vitro transcriptional activity than the wild-type, and the apparent weakening of the P1 promoter was roughly proportional to the length of the deleted AT-rich sequence. The implications of these findings for the possible in vivo role of the AT-rich region are discussed.