Nucleocytoplasmic shuttling of multiple signalling proteins is critical in the control of processes such as cell proliferation, differentiation, or apoptosis. One group of proteins whose activity depends on this nucleocytoplasmic traffic includes the mitogen-activated protein kinases. Usually, these kinases reside in the cytoplasm and move to the nucleus upon dual phosphorylation. One of these kinases, Erk5, has been found to reside in the nucleus of breast cancer cells that overexpress the ErbB2 receptor. This raises questions with respect to the mechanisms implicated in Erk5 nuclear location in these cells, as well as the biological consequences of this nuclear residency. In breast cancer cells overexpressing ErbB2, Erk5 dual phosphorylation required ErbB2 tyrosine kinase activity; however, Erk5 nuclear residency did not require ErbB2 activity. Furthermore, translocation of Erk5 from the cytosol to the nucleus occurred in the absence of dual phosphorylation. Nuclear residency of Erk5 in these cells depended on the integrity of a nuclear localization signal present in the unique C-terminus of Erk5. The Erk5 form expressed by these breast cancer cells included the N- and C-terminal cytoplasmic targeting signals, yet Erk5 was nuclear, and remained at this location throughout the interphase without being tightly bound to DNA. Biological studies using a mutant Erk5 that accumulates in the nucleus indicate that nuclear Erk5 favours MEF2-dependent transcriptional activity, and inhibits TRAIL-induced cell death.