Abstract Bacterial retroelements, or retrons, use reverse transcriptase (RT) to produce a multicopy single-stranded DNA (msDNA) molecule that is covalently linked to RNA. In these studies we show that a retron from Escherichia coli110, a clinical isolate, produces a novel RNA-less msDNA with a 5′ phosphate residue. The msDNA is a 74-nucleotide single-stranded DNA molecule with a stable stem–loop structure without a mismatched base pair. Only the genes encoding msDNA ( msd), msdRNA ( msr), and RT ( ret) are required to produce the msDNA molecule. The organization of these genes on the retron was similar to that of other elements producing branched msDNA–RNA. The conserved guanine, which is the branched residue in msDNA–RNA complexes and is essential for branch formation, is also present. Site-directed mutagenesis showed that this guanine is essential for the production of RNA-less msDNA. We postulate that the RNA-less msDNA in strain 110 is produced by nucleolytic cleavage of the branched msDNA–RNA compound.