The severe acute respiratory syndrome coronavirus (SARS-CoV) membrane protein is an abundant virion protein, and its interaction with the nucleocapsid protein is crucial for viral assembly and morphogenesis. Although the interacting region in the nucleocapsid protein was mapped to residues 168-208, the interacting region in the membrane protein and the interaction nature are still unclear. In this work, by using yeast two-hybrid and surface plasmon resonance techniques, the residues 197-221 of the membrane protein and the residues 351-422 of the nucleocapsid protein were determined to be involved in their interaction. Sequence analysis revealed that these two fragments are highly charged at neutral pH, suggesting that their interaction may be of ionic nature. Kinetic assays indicated that the endodomain (aa102-221) of the membrane protein interacts with the nucleocapsid protein with high affinity (K D = 0.55 ยฑ 0.04 ฮผM), however, this interaction could be weakened greatly by acidification, higher salt concentration (400 mM Na. Cl) and divalent cation (50 mM Ca2+), which suggests that electrostatic attraction might play an important role in this interaction. In addition, it is noted that two highly conserved amino acids (L218 and L219) in the membrane protein are not involved in this interaction. Here, we show that electrostatic interactions between the carboxyl termini of SARS-CoV membrane protein and nucleocapsid protein largely mediate the interaction of these two proteins. These results might facilitate therapeutic strategies aiming at the disruption of the association between SARS-CoV membrane and nucleocapsid proteins. ยฉ 2005 Elsevier Ltd.