5-Bromodeoxyuridine (BrdU) induces a phenomenon similar to cellular senescence in mammalian cells. AT-binding ligands such as Hoechst 33258 synergistically potentiate the effect of BrdU. We isolated mouse FM3A cell mutants resistant to Hoechst 33342 and characterized two highly resistant mutants. Two-dimensional gel electrophoresis followed by peptides sequence tags revealed that nuclear aldolase A was markedly increased in both mutants. Western blot analysis confirmed that nuclear aldolase A was increased leaving cytosolic aldolase A unaltered. Its mRNA levels were also increased in the mutants. Enforced expression of aldolase A conferred resistance to Hoechst 33342 on wild-type cells. Taken together, nuclear aldolase A was shown to somehow protect the cytotoxic effect of Hoechst 33342.