Prokaryotic toxin-antitoxin (TA) loci consist of two genes in an operon that encodes a metabolically stable toxin and an unstable antitoxin. The antitoxin neutralizes its cognate toxin by forming a tight complex with it. In all cases known, the antitoxin autoregulates TA operon transcription by binding to one or more operators in the promoter region while the toxin functions as a co-repressor of transcription. Interestingly, the toxin can also stimulate TA operon transcription. Here we analyse mechanistic aspects of how RelE of Escherichia coli can function both as a co-repressor and as a derepressor of relBE transcription. When RelB was in excess to RelE, two trimeric RelB(2)*RelE complexes bound cooperatively to two adjacent operator sites in the relBE promoter region and repressed transcription. In contrast, RelE in excess stimulated relBE transcription and released the RelB(2)*RelE complex from operator DNA. A mutational analysis of the operator sites showed that RelE in excess counteracted cooperative binding of the RelB(2)*RelE complexes to the operator sites. Thus, RelE controls relBE transcription by conditional cooperativity.