The structure and replication of a cold-adapted, temperature-sensitive (TS) mutant of an Asian (H2N2) influenza virus was compared with that of its wild-type (WT) parent. Viruses were grown in a chicken kidney cell system, and at the nonpermissive temperature of 40 C, production of infectious TS virus was about 100,000-fold less than at 35 C, in contrast to WT virus. Major structural polypeptides of each virus grown at 35 C were similar, except that the hemagglutinin glycopolypeptide (HA) of the TS virions was slightly more heterogenous than that of WT virions. Synthesis of viral polypeptides was examined by sodium dodecyl sulfate acrylamide gel electrophoresis of pulse-labeled infected cells. This revealed a defect in the synthesis of TS viral hemagglutinin that was most pronounced at the nonpermissive temperature. Other TS viral polypeptides appeared to be synthesized normally at 40 C. A defect in the TS virus hemagglutinin was also indicated by serological studies that demonstrated that TS virus hemagglutinin had lost antigenic sites present on the WT virus. Thus, it is concluded that the virus mutant examined contains lesions in the hemagglutinin gene, although the possibility of additional unrecognized lesions is not excluded.