Exonuclease I (ExoI) degrades single-stranded DNA in a 3' to 5'direction. It releases deoxyribonucleoside 5'-monophosphates in a stepwise manner and leaves 5'-terminal dinucleotides intact.

Used for primer removal from PCR mixtures, prior to PCR product sequencing, for one-tube 'megaprimer' PCR mutagenesis, removal of single-stranded DNA containing a 3'-hydroxyl terminus from nucleic acid mixtures, assay for the presence of single-stranded DNA with a 3'-hydroxyl terminus.