Abstract Insertion sequence IS 1 specifies the InsA, ΔInsA-B′-InsB and InsA-B′-InsB protein species. These three proteins have the identical α-helix-turn-α-helix motif that is likely to be responsible for DNA binding. In fact, InsA binds to the ends of IS 1, and regulates gene expression and transposition of IS 1. ΔInsA-B′-InsB and/or InsA-B′-InsB has been thought to possess a transposase-like activity. Here, I examined the actions of these proteins in vivo on the promoter (p insL) in the left end of IS 1. InsA repressed p insL-driven gene expression, both in cis and in trans. ΔInsA-B′-InsB inhibited it efficiently only when p insL was located near the construct where ΔInsA-B′-InsB is expressed. Furthermore, it has been shown that the possible −10 sequence of p insL is required for ΔInsA-B′-InsB to act on, but the −35 sequence where InsA binds specifically, is not. InsA-B′-InsB appeared not to work on a nearby p insL. The cis-action of ΔInsA-B′-InsB is consistent with the previous observation that the IS 1 transposase acts preferentially in cis. Interestingly, ΔInsA-B′-InsB acted on a nearby P3 promoter in the IS 1 insertion hotspot, and on another promoter outside the hotspot. ΔInsA-B′-InsB may generally interact with the regions in or around promoters owing to their low DNA helix stability. Note that IS 1 transposes preferentially into A + T-rich DNA segments, and that DNA is unwound from the −10 region of a promoter in transcription. The cis-preference of ΔInsA-B′-InsB would result in an overall reduction of transposition of IS 1 and its defective copy in a cell, allowing stable existence of the element in its bacterial host.