Three alkaline DNases, A, B, and C, with preference for the digestion of double-stranded DNA (dsDNA) were partially purified from microplasmodia of Physarum polycephalum. They were very similar but differed in their isoelectric points. These were pH 5.8 for DNase A, 7.1 for DNase B, and 9.1 for DNase C. All three enzymes consisted of a single polypeptide chain with a molecular weight of 16,000 to 17,000, which readily formed high molecular weight complexes with low enzyme activity. These complexes could be reversibly dissociated by urea, and DNase activity was quantitatively reactivated. The DNases hydrolyzed the substrate DNA by an endonucleolytic mechanism which gave 5'-phosphorylated products. Divalent cations, MnCl2 or MgCl2, were essential for enzyme activity at the optimum pH of approximately 8.5 and at low ionic strength. The optimal conditions of pH, buffer, divalent cations and ionic strength and the extent of inhibition by salt, phosphate ions or urea differed slightly but significantly between the different isoenzymes.