A double-stranded RNA (dsRNA)-specific modification activity from Xenopus oocytes and human cells dsRNA modifier) converts adenosine residues present in dsRNA to inosines. The function of the dsRNA modifier is unknown, although it has been suggested that it may be part of the cellular antiviral response. We investigated the relationship between the activity of the dsRNA modifier, viral infection, and the antiviral response in human cells induced by poly(rI)-poly(rC) [poly(I.C)] treatment. We found, unexpectedly, that treatment of HeLa cells with poly(I.C) or other dsRNA molecules resulted in the dramatic inhibition of the dsRNA modifier. Mixing experiments, reconstruction experiments, and pretreatment of extracts with RNases indicated that inhibition of the dsRNA modifier did not result from the continued presence of a soluble inhibitor such as dsRNA) in the in vitro modification reactions. Treatment of cells with cyclohexamide or dactinomycin simultaneously with the poly(I.C) demonstrated that in vivo inhibition of the dsRNA modifier did not require new transcription or translation. The dsRNA modification activity was also substantially inhibited in cells infected with poliovirus and was slightly inhibited in cells infected with adenovirus. The inhibition of the dsRNA modifier during the antiviral state is thus not consistent with an antiviral function, and instead suggests another cellular function for dsRNA modification.