Expression of the lacZ gene from the supercoiling-sensitive leu-500 promoter on a plasmid in topA mutant cells was stimulated by activating a divergently oriented Tac promoter, 400 bp upstream from leu-500. The stimulation was approximately threefold regardless of whether the Tac promoter drove the expression of the tet gene, whose product is membrane bound, or of the cat gene, whose product is cytosolic. Putting a second copy of the Tac promoter downstream from lacZ, approximately 3,000 bp from leu-500 in the same orientation as the latter, resulted in 30-fold increase in lacZ expression upon isopropyl-beta-D-thiogalactopyranoside induction. Again, these effects were independent of the nature of the gene upstream from leu-500 (tet or cat). With both tet- and cat-harboring constructs, activation of the two Tac promoter copies caused plasmid DNA to become hypernegatively supercoiled in topA mutant cells. Thus, neither leu-500 activation nor hypernegative plasmid DNA supercoiling appears to require membrane anchoring of DNA in this system. Replacing the downstream copy of Tac with a constitutive promoter resulted in high-level lacZ expression even when the upstream copy was repressed. Under these conditions, no hypernegative DNA supercoiling was observed, indicating that the activity of plasmid-borne leu-500 in topA mutant cells does not necessarily correlate with the linking deficit of plasmid DNA. The response of the leu-500-lacZ fusion to downstream transcription provides a sensitive assay for transcriptional supercoiling in bacteria.