Due to their ability to degrade RNA, selected members of the bovine pancreatic ribonuclease A (RNase A) superfamily are potent cytotoxins. The cytotoxic action requires the interaction of the enzyme with the cellular membrane, its internalization and translocation to the cytosol, and the degradation of ribonucleic acid whereby they cause cell death. The interplay of these processes as well as the role of the thermodynamic and proteolytic stability, the catalytic activity, and the evasion from the intracellular ribonuclease inhibitor (RI) has not yet been fully elucidated. In vitro, the catalytic activity of most RNases, however, is abolished by RI, which might derogate their applicability as cytotoxins. Consequently, the development of RNase derivatives with the ability to evade RI binding is a desirable goal. In this work, tandem enzymes consisting of two RNase A units that are covalently bound via a peptide linker were generated by gene duplication. As deduced from the alignment of the crystal structure of the tandem enzymes with that of the RI•RNase A complex, one RNase A unit of the tandem enzyme can still be bound by RI. The other unit, however, should remain unbound because of steric hindrance. This free RNase A unit was expected to maintain its activity and to act as a cytotoxic agent. Despite a complete in vitro inhibition by RI, tandemization was found to endow RNase A with remarkable cytotoxic activity. While monomeric RNase A is not cytotoxic, IC50 values of the RNase A tandem variants of 70.3-12.9 µM were determined. Hence, the RI sensitivity of the tandem enzymes was apparently surmounted by an enhanced endocytosis efficiency.