Guinea pig epidermal DNAase I was purified from an epidermal extract by a procedure including DEAE-cellulose chromatography, Sephadex G-100 gel filtration and Con A-Sepharose affinity chromatography. The purified enzyme contained no detectable activities of acid DNAase, alkaline RNAase, phosphodiesterase or acid or alkaline phosphatase, but was contaminated with acid RNAase activity. The molecular weight of the enzyme was estimated to be 33 000 by sucrose density gradient centrifugation and Sephadex G-100 gel filtration. Its isoelectric point is 5.2 +/- 0.1. The enzyme requires divalent cations and exhibits two pH optima that are dependent on divalent cations: in the presence of Mn2+, the optimum pH is about 7.5 in 50 mM Tris-HCl buffer and in the presence of Mn2+, the pH is 6.4 in 50 mM cacodylate-HCl buffer. The enzyme hydrolyzes native DNA about 6-times faster than denatured DNA, producing 5'-phosphoryl and 3'-hydroxyl terminated oligonucleotides with an average chain length of about eight nucleotides, and converts double-stranded and circular DNA to relaxed and linear forms. The enzyme is inhibited by G-actin and antiserum against bovine pancreatic DNAase A. Thus this enzyme is classified as DNAase I.