A DNase, designated Exonuclease II, has been purified 2,000-fold from whole cell extracts of bakers' yeast. It degrades single-stranded but not double-stranded DNA. The enzyme has a pH optimum around 8 and requires at least 2 mM Mg2+ for activity. It is slightly stimulated by dithiothreitol and inhibited by N-ethylmaleimide, suggesting that --SH groups are essential for activity; heparin also inhibits the activity. With 0.1 enzyme unit the Km has been determined to 4 nM of DNA ends and Vmax to 0.52 pmol of liberated nucleotides per min. The Mr is around 120,000. The enzyme does not degrade circular single-stranded M13 DNA, whereas linearized M13 DNA is degraded. The products are 5'-deoxyribomononucleotides exclusively. Using 5'-end labeled and 3'-end labeled DNA fragments as substrates, partially degraded DNA is only detectable in the latter case, showing that the exonuclease solely attacks DNA from the 5'-end. This distinguishes Exonuclease II from other exonucleases degrading DNA from the 5'-end, since they all either produce a mixture of 5'-mono- and oligonucleotides or 3'-mononucleotides. Analysis of 3'-end labeled fragments after incubation shows that the rate of exonucleolytic cleavage was dependent on the DNA sequence at the 5'-end.