Necdin is a growth suppressor expressed predominantly in postmitotic neurons. The necdin gene is involved in the etiology of the genomic imprinting-associated neurodevelopmental disorder Prader–Willi syndrome and belongs to the MAGE gene family. All the MAGE family proteins contain a large homology domain termed the MAGE homology domain (MHD). We here characterize the regions of necdin required for the protein–protein interaction, nuclear matrix targeting, and cell growth suppression. The region including entire MHD (amino acids 116–280) of necdin was required for its interaction with p53, while the regions amino acids 144–184 and 191–222 within the MHD were required for both the nuclear matrix targeting and the cell growth suppression of osteosarcoma SAOS-2 cells. The amino-terminal proline-rich acidic region (amino acids 60–100) was also necessary for cell growth suppression. Tetracycline-regulatable overexpression of necdin induced growth arrest of SAOS-2 cells in a reversible manner, and the necdin-overexpressing cells showed a large, flattened morphology with double nuclei. In contrast, a necdin mutant lacking amino acids 191–222 did not induce such changes. These findings suggest that different functions of necdin are mediated via its distinct domains. © 2004 Wiley-Liss, Inc.