The temperature sensitivity, level of attenuation, and phenotypic stability of an influenza B/Texas/1/84 X B/Ann Arbor/1/66 cold-adapted (ca) reassortant virus that received the RNA segments that encode the hemagglutinin and neuraminidase surface glycoproteins from the B/Texas/84 wild-type virus and the remaining six RNA segments from the B/Ann Arbor/66 ca donor virus were evaluated. In comparison to wild-type virus, the ca reassortant was restricted in replication in the upper and lower respiratory tracts of hamsters and chimpanzees. This clearly demonstrated that the six transferable RNA segments of the B/Ann Arbor/66 ca donor virus specify the attenuation phenotype for animals with a 37 degrees C core body temperature. In addition, the ca virus retained the temperature-sensitive (ts) phenotype after replication in hamsters and chimpanzees. To further evaluate its phenotypic stability, the ca reassortant virus was isolated after 6-15 d of replication in hamsters that were immunosuppressed with cyclophosphamide, and the isolates were tested for their temperature sensitivity in vitro and for their level of replication in immunocompetent hamsters. The isolated viruses retained their ts and attenuation phenotypes even after prolonged replication in vivo. Thus, the B/Ann Arbor/66 ca donor virus can transfer the desired properties of attenuation and phenotype stability to its reassortants. The findings support the continued evaluation of ca reassortant influenza B virus vaccines in humans, including fully susceptible children.