By a heterotypic cross between influenza viruses A/WSN (H1N1) and B/Yamagata/1/73, we obtained a mutant of B/Yamagata (AWBY-234), which expressed a greatly truncated NS1 protein with molecular weight of 13,500. Direct sequencing of the NS gene of the mutant revealed a deletion of a single uridine base at the position 310, 311, or 312 of the plus sense RNA, giving rise to a new stop codon at the position 314-316. The resulting NS1 protein was predicted to be composed of only 90 amino acids, compared with 281 with the NS1 of the wild-type B/Yamagata. AWBY-234 grew normally and induced a typical cytopathic effect in infected MDCK cells much earlier after infection than did the wild-type B/Yamagata. A single gene reassortant in which the NS gene of AWBY-234 was transferred to B/Lee/40 inherited these characteristics from AWBY-234 parent. The single gene reassortant, but not the control reassortant, contained a significant amount of defective particles which can complement each other to produce infectious virus.