Human DNase I, an enzyme used to treat cystic fibrosis patients, has been engineered to more effectively degrade double-stranded DNA to lower molecular weight forms by introducing positively charged amino acids at positions that can interact favorably with the proximal negatively charged phosphate groups of the DNA. A series of combination mutants having from one to six additional basic residues compared with the wild type has been constructed, expressed in human 293 cells, and characterized. The degree of hyperactivity for the mutants was highly dependent upon the conditions in various assays, including the concentration and length of the DNA substrate and the salt and divalent metal ion concentrations. The level of hyperactivity was inversely proportional to both DNA concentration and DNA length, consistent with the processive nicking mechanism for the hyperactive variants. Salt was inhibitory for wild type DNase I but actually enhanced the activity of the hyperactive variants. Under optimal conditions for wild type, variants with one additional positive charge possessed the highest activity, which was only severalfold greater than that for wild type. However, in the presence of low DNA concentrations and molecular weights, no Ca2+, and 150 mM NaCl, the variant with six engineered basic residues was most active, having >10,000-fold higher activity than the wild type enzyme. Therefore, any potential increase in potency for the hyperactive variants in vivo will be determined by the concentration, length, and environment of the DNA.