Bacillus pumilus ribonuclease (binase) exhibits cytotoxic and oncolytic properties, while causing genotoxic effects at high concentrations. Mutants that have reduced catalytic activity and preserve the antitumor properties of the native enzyme could exert lower toxic side effects. Mutant binase forms with the Lys26Ala and His101Glu single substitutions were obtained by site-directed mutagenesis. A comparative analysis of Escherichia coli- and Bacillus subtilis-based expression systems demonstrated that the latter is better to use to produce the binase mutants. The binase mutants with reduced catalytic activity were isolated and purified to homogeneity by ion exchange chromatography; the maximum yield was 25 mg/L. Catalytic activities of the mutants toward natural RNA-substrates in comparison with those for native binase were estimated at 11% and 0.02%, respectively. Like native binase, the Lys26Ala mutant was found to be cytotoxic to the A549, BT-20, and HuTu 80 tumor cell lines, but did not substantially affect normal WI-38 cells. The His101Glu mutant did not show cytotoxicity.