The anti-prohferative function of the p53 tumour suppressor depends primarily on its ability to act as a transcription factor and to coordinate the cellular response to various stress signals. p53 can induce several cellular responses, including cell-cycle arrest, DNA repair and apoptosis. The choice of the p53 response is thought to depend on many factors, including the action of p53-cooperating molecules. The cofactor JMY has been demonstrated to cooperate with the p300 coactivator to augment p53 activity. The aim of this study was to investigate the mechanisms regulating JMY function to further understand its role in the stress response. Results presented here indicate that the subcellular localisation of JMY is regulated by the cellular environment. Whilst DNA damage stimulated the translocation of JMY from the cytoplasm to the nucleus, removal of proliferative and survival signals by serum deprivation induced rapid nuclear export of JMY and compromised its ability to affect cell cycle progression and stimulate apoptosis. The Mdm2 oncoprotein negatively regulates p53 activity at multiple levels including regulation of the intracellular location of p53 and inhibition of its interaction with the transcriptional coactivator CBP. Mdm2 interacts with JMY in cells, and enhances its cytoplasmic localisation without promoting its degradation. Leptomycin B blocked Mdm2-dependent redistribution of JMY, implying the involvement of a nuclear export mechanism. DNA damage was found to abrogate the effect of Mdm2 on the subcellular localisation of JMY, and to reduce the interaction between the two proteins. Mutations in the ubiquitin ligase domain of Mdm2, as well as removal of the proline-rich region in JMY abolished the Mdm2- mediated cytoplasmic translocation of JMY. These data suggest the existence of a functional relationship between the p53 cofactor JMY, and its major inhibitor Mdm2, which is likely to impact on p53 activity. Importantly, regulation of the intracellular localisation of JMY might provide an additional mechanism of modulating the p53 response.