The most temperature-sensitive dsRNA-negative mutant of reovirus serotype 3 is ts447; the amount of dsRNA formed in cells infected with it at 39 degrees is less than 0.1% of that formed in cells with wt virus at 37 degrees. The genome segment in which this mutation is located is S2. We compare here the sequence of the S2 genome segment of wt reovirus serotype 3 with that of mutant ts447. The two sequences differ in three locations, at two of which there are C to U transitions, while at the third there is an A to G transition. All cause amino acid changes (Ala to Val, Ala to Val, and Asn to Asp, respectively). One mutation (at nucleotide position 581, which causes an Ala to Val change) causes the length of an alpha-helix to be significantly reduced and may be that which is responsible for the ts phenotype.