Toxin-antitoxin (TA) systems on the chromosomes of free-living bacteria appear to facilitate cell survival during intervals of stress by inducing a state of reversible growth arrest. However, upon prolonged stress, TA toxin action leads to cell death. They have been implicated in several clinically important phenomena--bacterial persistence during antibiotic treatment, biofilm formation and bacterial pathogenesis--and serve as attractive new antibiotic targets for pathogens. We determined the mode of action of the YafQ toxin of the DinJ-YafQ TA system. YafQ expression resulted in inhibition of translation, but not transcription or replication. Purified YafQ exhibited robust ribonuclease activity in vitro that was specifically blocked by the addition of DinJ. However, YafQ associated with ribosomes in vivo and facilitated rapid mRNA degradation near the 5' end via cleavage at AAA lysine codons followed by a G or A. YafQ(H87Q) mutants lost toxicity and cleavage activity but retained ribosome association. Finally, LexA bound to the dinJ-yafQ palindrome and triggered module transcription after DNA damage. YafQ function is distinct from other TA toxins: it associates with the ribosome through the 50S subunit and mediates sequence-specific and frame-dependent mRNA cleavage at (5')AAA-G/A(3') sequences leading to rapid decay possibly facilitated by the mRNA degradosome.