Using a reovirus reverse genetics system, we have identified the 5' sequences required of an engineered s2 ssRNA for efficient incorporation into the dsRNA genome of Reovirus. Employing an engineered, functionally active reovirus S2/CAT gene retaining the first 198 5' terminal nucleotides and the last 284 3' terminal nucleotides of the wild-type S2 segment, we have determined the 5' sequence required by a ssRNA to be recognized, replicated to dsRNA, and stably incorporated into an infectious reovirus. The 5' sequence retains 96 nucleotides of the wild-type s2 ssRNA and a predicted sequence-structure element. Within these 96 nucleotides, we have identified three nucleotides A-U-U at positions 79-81 that are essential for the incorporation of in vitro-generated ssRNAs into new reovirus progeny viral particles. This study establishes a firm foundation for additional investigation into the assortment and encapsidation mechanism of all 10 ssRNAs into the dsRNA genome of reovirus.