The MDMX gene product is related to the MDM2 oncoprotein, both of which interact with the p53 tumor suppressor. A novel transcript of the MDMX gene has been previously identified that has a short internal deletion of 68 base pairs, producing a shift in the reading frame after codon 114, resulting in the inclusion of 13 novel amino acids (after residue 114) followed by a stop codon at amino acid residue 127. This truncated MDMX protein, termed MDMX-S, represents only the p53 binding domain and binds and inactivates p53 better than full-length MDMX or MDM2. Here we show that when expressed in cells, MDMX-S is targeted more efficiently to the nucleus than MDMX. MDMX-S suppresses p53-mediated transcription from a p53 target promoter better than full-length MDMX. The DNA damage inducibility of these p53 responsive promoters was suppressed better by MDMX-S than by MDMX. Analysis of the MDMX-S protein indicated that the 13 novel amino acids at its carboxy terminus was responsible for high affinity binding to p53 in vitro and for high level expression of the protein in cells. Deletion of this 13 amino acid sequence resulted in a protein that was not able to bind p53 and was not able to be expressed well in cells. Taken together, these data point to an important domain within MDMX-S that enables it to function well in vivo to block p53 activity. Published 2003 Wiley-Liss, Inc.