The genomes of viruses in the family Reoviridae consist of segmented double-stranded RNA. There are 10 to 12 segments depending on the genus. The 5' ends and the 3' ends of the RNAs present conserved motifs for each virus genus. These conserved motifs have been hypothesized to play a role in genomic segment assortment during virus morphogenesis. Using a set of monoclonal antibodies we have tried to identify rotaviral proteins that bind to RNA during infection in cell culture. This methodology takes advantage of being able to label RNA in vitro to high specific activity and also of solid phase processing of RNA-protein complexes. After cross-linking the RNA to protein in infected cells, protein-RNA complexes are precipitated with a specific MAb; then, the RNA in the complex is labeled in vitro and the protein or nucleic acid moieties are analyzed by usual protocols. This paper describes results using an anti NSP3 MAb. In infected cells, we have shown that NSP3 binds to the eleven messenger RNAs, and that a sequence from nucleotides 8 to 15 is protected fi-om digestion with RNAse T1 by NSP3 in the RNA-protein complex. The availability of recombinant protein NSP3 expressed in the baculovirus-insect cell system has allowed the sequence specificity of NSP3 to be studied in vitro. The minimal sequence recognized by NSP3 is GACC. The role of NSP3 in rotavirus replication is discussed based on these results and by comparison with other RNA-binding proteins of members of the Reoviridae family.