Retron reverse transcriptases are unusual procaryotic enzymes capable of synthesis of low-molecular-weight DNA by reverse transcription. All of the so-far-described DNA species synthesized by retron reverse transcriptases have been identified as multicopy single-stranded DNA. We have shown that Salmonella enterica serovar Enteritidis is also capable of synthesis of the low-molecular-weight DNA by retron reverse transcriptase. Surprisingly, Salmonella serovar Enteritidis-produced low-molecular-weight DNA was shown to be a double-stranded DNA with single-stranded overhangs (sdsDNA). The sdsDNA was 72 nucleotides (nt) long, of which a 38-nt sequence was formed by double-stranded DNA with 19- and 15-nt single-stranded overhangs, respectively. Three open reading frames (ORFs), encoded by the 4,053-bp plasmid, were essential for the production of sdsDNA. These included an ORF with an unknown function, the retron reverse transcriptase, and an ORF encoding the cold shock protein homologue. This plasmid was also able to confer phage resistance onto the host cell by a mechanism which was independent of sdsDNA synthesis.