When SI nuclease from Takadiastase was partially purified according to previously reported methods, it showed a 10 to 15 fold increase in specific-activity. Although such preparations were highly active on single-stranded DNA, they had traces of activity on native DNA and were contaminated by TI-RNase. The SI enzyme was further purified by a single step of affinity chromatography on single-stranded DNA-acrylamide column to a final purification of 275-fold. This preparation was free of Tl-RNase and had an absolute specificity for single-stranded DNA.