Addition of double-stranded RNA (dsRNA) to extracts of interferon-treated HeLa cells results in the synthesis of 2’,8-oligo(A) from ATP and in the phosphorylation of a ribosome-associated protein of M, = 72,000. Previously described assays were used to investigate the structural requirements of dsRNA for the activation of these two enzymatic activities. Poly(CG) with different ratios of C/G was synthesized with polynucleotide phosphorylase. These polynucleotides were either annealed with poly(1) to form mismatched dsRNA or digested with ribonuclease T1 to produce smaller polynucleotides. Polymers with an average of one mismatch every eight nucleotides failed to activate the 2’,5’-oligo(A) polymerase and protein kinase, whereas polymers with a mismatch every 45 nucleotides were fully active. The polynucleotides obtained by T1 digestion of poly(CG) were fractionated by gel filtration into discrete size polymers. These sized polynucleotides were annealed with high molecular weight poly(1) and assayed for activation of 2’,5’-oligo(A) polymerase and protein kinase. These enzymes could not be activated by dsRNA containing poly(C) shorter than 30 nucleotides. Maximal activation was obtained with dsRNA containing poly(C) longer than 65 to 80 nucleotides. A similar size requirement for activation was observed with dsRNA formed with poly(A) and poly(U) of known length. These results indicate that a relatively long stretch of base pairs, uninterrupted by either a mismatch or a discontinuity in one of the complementary strands, is required for the activation of the two enzymes studied. These structural characteristics are similar to those previously shown to be required for the induction of interferon by dsRNA.