Influenza B virus BM2 is a type III integral membrane protein that displays H(+) ion-channel activity. Analysis of BM2-knockout mutants has suggested that this protein is a necessary component for the capture of M1-viral ribonucleoprotein (vRNP) complex at the plasma membrane and for incorporation of vRNP complex into the virion during the assembly process. BM2 comprises 109 amino acid residues and possesses a longer cytoplasmic tail than the other 3 integral membrane proteins (hemagglutinin, neuraminidase, and NB). To explore whether the cytoplasmic tail of BM2 is important for infectious virus production, a series of BM2 deletion mutants lacking 3-9 amino acid residues at the carboxyl-terminus, BM2Delta 107-109, BM2Delta 104-109 and BM2Delta 101-109, were generated by reverse genetics. Intracellular transport and incorporation into virions were indistinguishable between truncated BM2 proteins and wild-type BM2. BM2Delta 107-109 mutant produced similar levels of infectious virus to wild-type virus and displayed spherical morphology. However, BM2D 104-109 and BM2Delta 101-109 mutants produced viruses containing dramatically reduced vRNP complex as in BM2-knockout mutants and formed enlarged, irregularly shaped virions. Moreover, gradient separation of membranes indicated that membrane association of M1 from mutants was greatly affected by carboxyl-terminal truncations of BM2. Studies of alanine substitution mutants further suggested that amino acid sequences in the 98-109 region are variable, while those in the 86-97 region are prerequisite for innate BM2 function. These results indicate that the cytoplasmic domain of BM2 protein is required for firm association of M1 protein with lipid membranes, vRNP complex incorporation into virions and virion morphology.