We have previously described a human pancreatic-ribonuclease variant, named PE5, which carries a non-contiguous extended bipartite nuclear localization signal. This signal comprises residues from at least three regions of the protein. We postulated that the introduction of this signal in the ribonuclease provides it with cytotoxic activity because although the variant poorly evades the ribonuclease inhibitor in vitro, it is routed to the nucleus, which is devoid of the inhibitor. In this work, we have investigated the relationship between the cytotoxicity produced by PE5 and its ability to reach the nucleus. First, we show that this enzyme, when incubated with HeLa cells, specifically cleaves nuclear RNA while it leaves cytoplasmic RNA unaffected. On the other hand, we have created new variants in which the residues of the nuclear localization signal that are important for the nuclear transport have been replaced. As expected, the individual changes produce a significant decrease in the cytotoxicity of the resulting variants. We conclude that the nuclear transport of PE5 is critical for its cytotoxicity. Therefore, routing a ribonuclease to the nucleus is an alternative strategy to endow it with cytotoxic activity.