The acid deoxyribonucleases [DNase II; EC 3.1.4.6] in human urine were purified approximately 400- to 500-fold by phosphocellulose chromatography, gel filtration on Sephadex G-75 and isoelectric focusing, with a total recovery of 22%. The enzymes were present in a least three forms with different isoelectric points, pHs 6.4, 6.6, and 6.8. However, other properties were essentially similar. The enzymes did not require divalent cations for activity, and the optimal pHs were at 5.1 to 5.3 in 33 mM acetate buffer. They had a molecular weight of around 36,000, as estimated by gel filtration on Sephadex G-75. The enzymes were endonucleases which hydrolyzed native, double-stranded DNA about 5 to 15 times faster than thermally denatured DNA. The products formed from native DNA were 3'-phosphoryl- and 5'-hydroxy-terminated oligonucleotides. The average chain length of the limit digests with these enzymes was approximately 11 to 15, and the major fragments were longer than pentanucleotides. The final preparations were free of nonspecific acid and alkaline phosphatases and phosphodiesterase, but contained contaminating ribonuclease activity.