MazF is an Escherichia coli toxin that is highly conserved among the prokaryotes and plays an important role in growth regulation. When MazF is induced, protein synthesis is effectively inhibited. However, the mechanism of MazF action has been controversial. Here we unequivocally demonstrate that MazF is an endoribonuclease that specifically cleaves mRNAs at ACA sequences. We then demonstrate its enzymatic specificity using short RNA substrates. MazF cleaves RNA at the 5'-end of ACA sequences, yielding a 2',3'-cyclic phosphate at one side and a free 5'-OH group at the other. Using DNA-RNA chimeric substrates containing XACA, the 2'-OH group of residue X was found absolutely essential for MazF cleavage, whereas all the other residues may be deoxyriboses. Therefore, MazF exhibits exquisite site specificity and has utility as an RNA-restriction enzyme for RNA structural studies or as an mRNA interferase to regulate cell growth in prokaryotic and eukaryotic cells.