The affinity of the reovirus sigma 3 protein for double-stranded RNA (dsRNA) is well established, and efforts have been made to identify the amino acids involved in this property. In the present study, we further examined the importance of two basic amino acids motifs, located in the carboxy-terminal third of the protein. Mutants, previously characterized in COS cells, were expressed in bacterial cells using the pET expression system. The capability of the different mutants to interact with dsRNA was then determined by the binding of radiolabeled dsRNA to proteins resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. It appears that the most carboxy-terminal motif is absolutely required for the binding but the second motif also contributes to this property. However, only the carboxy-terminal motif is required for normal binding upon removal of the amino-terminal domain of the protein by proteolytic cleavage, a procedure previously shown to increase dsRNA-binding. The basic charges in both motifs are important, while breaking of their potential to adopt an alpha helical configuration does not affect binding efficiency. Furthermore, alanine substitution of a single basic amino acid in the carboxy-terminal motif can be sufficient to strongly reduce the binding of dsRNA to the protein. Altogether, these data suggest that basic amino acids of the sigma 3 carboxy-terminal motif are directly involved in dsRNA binding, while the other basic motif may contribute by preventing an inhibitory effect of the amino-terminal portion of the protein.