Purified by chromatography. Spleen exonuclease (Phosphodiesterase II) hydrolyzes 3'-phosphomononucleotides from oligonucleotides containing a 5'-hydroxyl terminus. The enzyme's specificity for the 5'-phosphate end of a polynucleotide chain after conversion to 5'-OH by use of E. coli alkaline phosphatase makes spleen exonuclease very important in the characterization of polynucleotide chain length, base composition and identity of terminal nucleotide. Spleen exonuclease splits off, sequentially, nucleoside 3'-phosphates, starting at the 5'-OH end, from ribo-or deoxyribo-oligonucleotides. RNA ``core'', alkali-treated DNA, poly(A), poly(U), and poly(I) are acted upon. Prior removal of the 5'-terminal phosphate group is essential. Unit Definition: 1 unit increases the absorbance at 260 nm by 0.200 in 30 minutes at 37°C, pH 6.5, with an RNA Core as substrate. Optimum pH: 5.5 using succinate and phosphate buffer and an acid DNase digest; pH 6-7 with 0.1M acetate buffer. Activity: ≥10 units per