An immunochemical binding assay was used to investigate the reactivity of radioactively labeled viral RNAs from poliovirus-infected cells with antibodies to the synthetic double-stranded RNA, poly(I) poly(C). A RNase-free antibody-containing serum fraction was employed. Poliovirus replicative form reacted with the antibodies to poly(I) .poly(C) as well as or better than poly(I) .poly(C). Poliovirus replicative intermediate reacted with the antibodies to a greater extent than poliovirus single-stranded RNA, but both were less reactive than replicative form. The use of the immunochemical binding assay with sucrosegradient fractions demonstrated that for both poliovirus single-stranded RNA and replicative form the peak of reactivity with the antibodies was coincident with the peak of radioactive material precipitated by trichloroacetic acid. The proportion of replicative intermediate that reacted with the antibody increased in sucrose-gradient fractions containing the more slowly sedimenting RI RNA.