A new exonuclease of Escherichia coli K12, exonuclease VII, has been purified 1700-fold and characterized. The enzyme is specific for single-stranded DNA and can initiated hydrolysis at both 5' and 3' termini. It is also capable of thymine-dimer excision in vitro. The limit products of the reaction are oligonucleotides, predominantly in the range of tetramers to dodecamers. DNA is hydrolyzed by the enzyme in a processive fashion. Mutants of E. coli have been isolated having reduced levels of exonuclease VII activity in crude extracts. Mapping studies place the exonuclease VII locus between 45 and 56 minutes on the E. coli K12 linkage map.