Cold adapted (ca) B/Ann Arbor/1/66 is the master donor virus for the influenza B (MDV-B) vaccine component of the live attenuated influenza vaccine (FluMist). The six internal genes contributed by MDV-B confer the characteristic cold-adapted (ca), temperature-sensitive (ts) and attenuated (att) phenotypes to the vaccine strains. Previously, it has been determined that the PA and NP segments of MDV-B control the ts phenotype while the att phenotype requires the M segment in addition to PA and NP. Here, we show that the PA, NP and PB2 segments are responsible for the ca phenotype of MDV-B when examined in chicken cell lines. Five loci in three RNA segments, R630 in PB2, M431 in PA and A114, H410 and T509 in NP, are sufficient to allow efficient virus growth at 25 degrees C. Substitution of these five amino acids with wt (wild type) residues completely reverted the MDV-B ca phenotype. Conversely, introduction of these five ca amino acids into B/Yamanashi/166/98 imparted the ca phenotype to this heterologous wt virus. In addition, we also found that the MDV-B M1 gene affected virus replication in chicken cells at 33 and 37 degrees C. Recombinant viruses containing the two MDV-B M1 residues (Q159, V183) replicated less efficiently than those containing wt M1 residues (H159, M183) at 33 and 37 degrees C, implicating the role of the MDV-B M segment to the att phenotype. The complexity of the multigenic signatures controlling the ca, ts and att phenotypes of MDV-B provides the molecular basis for the observed genetic stability of the FluMist vaccines.