The mature flavivirus particle comprises a nucleocapsid core surrounded by a lipid bilayer containing the membrane (M) (derived from the precursor prM) and envelope (E) proteins. The formation of intracellular prM/E heterodimers occurs rapidly after translation and is believed to be important for the assembly and secretion of immature virus particles. In this study, the role of the His residue at position 39 in the M protein (M39) of dengue virus type 2 (DENV-2) in the virus life cycle was investigated. Mutations encoding basic (Arg), non-polar (Leu and Pro) and uncharged polar (Asn, Gln and Tyr) amino acids at M39 were introduced into a DENV-2 genomic-length cDNA clone and their effects on virus replication were examined. Substitution of the His residue with non-polar amino acids abolished virus replication, whereas substitution with basic or uncharged polar amino acids decreased virus replication moderately ( approximately 2 log(10) p.f.u. ml(-1) decrease in viral titre for Arg and Asn) or severely (>3.5 log(10) p.f.u. ml(-1) decrease in viral titre for Gln and Tyr). Selected mutations were introduced into a prM-E gene cassette and expressed transiently in COS cells to investigate whether the mutations impaired prM/E association or secretion. None of the mutations was found to disrupt the formation of intracellular prM/E heterodimers. However, the mutations that abolished virus replication prevented secretion of prM/E complexes. The results of this study pinpoint a critical residue in the M protein that potentially plays a role in viral morphogenesis, secretion and entry.