The synthetic interferon (IF) inducer rIn.rCn was modified by substituting the ribosyl residues with either their corresponding deoxy(dC or dI) or 2'-O-methyl analogues (mC or mI). The polynucleotide duplexes of these analogues are inactive as IF inducers. Circular dichroism (CD) studies revealed that, while the deoxy substitution produces significant changes in the conformation of rIn.rCn, the 2'-O-methyl substitution produces no detectable change. Biological competition experiments indicated that the methylated duplexes mIn.rCn, rIn.mCn, and mIn.mCn all compete with rIn.rCn for IF induction, while the deoxy duplexes dIn.rCn and rIn.dCn do not. These results are consistent with those predicted from the CD data. Copolymer duplexes (mI,rI)n.(mC,rC)n of varying degrees of methylation and residue clustering were also evaluated for IF induction in human fibroblasts. The IF-inducing capabilities of these duplexes correlated highly with the presence of clusters containing six or more consecutive ribosyl residues. These combined observations suggest that interaction of rIn.rCn with the cell in the induction process may occur in a biphasic manner involving first the topological recognition of a large segment of the RNA to allow for proper binding to the putative cellular receptor, followed by recognition of a much smaller region of the RNA corresponding to 6--12 consecutive ribosyl residues (0.5 to 1 helical turn) which is responsible for the triggering of the induction process.