Based on the antiviral effect of interferon on rotavirus replication the inhibitory effect of 2',5'-oligoadenylates on MRNA and double-stranded RNA synthesis was studied using an in vitro assay. The chemically synthesized oligonucleotides were used to determine several characteristics of the inhibitory effect, such as chain length, presence of phosphate residues at the 5'-end, and the 2',5'-phosphodiester bond itself. In vitro transcription was inhibited by oligos with 5 or more adenine residues at a final concentration of 100 microM or greater. This result makes rotavirus transcriptase different from other viruses in which the inhibitory effects are associated with dinucleotides and trinucleotides. The inhibitory effect was increased when the oligo contained a phosphate residue at the 5'-end; in this case, inhibition was also seen at lower oligo concentrations as well as at shorter oligo chain length. The study of the kinetics of inhibition showed that the inhibition by p(A2'p5')(3)3A was competitive with a Ki value of 256 microM. The effect of the oligonucleotides on the in vitro viral RNA replication showed that the 2',5'-oligoadenylates were not able to significantly inhibit the in vitro rotavirus RNA synthesis. The lack of inhibition in the in vitro assay was very peculiar since RNA transcription and replication involves the viral RNA polymerase, VP1.