Genome sequence analyses of Escherichia coli K-12 revealed four copies of long repetitive elements. These sequences are designated as long direct repeat (LDR) sequences. Three of the repeats (LDR-A, -B, -C), each approximately 500 bp in length, are located as tandem repeats at 27.4 min on the genetic map. Another copy (LDR-D), 450 bp in length and nearly identical to LDR-A, -B and -C, is located at 79.7 min, a position that is directly opposite the position of LDR-A, -B and -C. In this study, we demonstrate that LDR-D encodes a 35-amino-acid peptide, LdrD, the overexpression of which causes rapid cell killing and nucleoid condensation of the host cell. Northern blot and primer extension analysis showed constitutive transcription of a stable mRNA (≈ 370 nucleotides) encoding LdrD and an unstable cis-encoded antisense RNA (≈ 60 nucleotides), which functions as a trans-acting regulator of ldrD translation. We propose that LDR encodes a toxin–antitoxin module. LDR-homologous sequences are not present on any known plasmids but are conserved in Salmonella and other enterobacterial species.