A monoclonal antibody highly specific for (2'-5')adenylyladenosine oligonucleotides was used together with a 125I-labeled analog of this compound to detect and quantify phosphorylated and nonphosphorylated (2'-5')adenylyladenosine oligonucleotides in a variety of tissues and cells. These oligonucleotides were first assayed as a whole in perchloric acid extracts and then further individually characterized by HPLC analysis. Their sensitivity to alkaline phosphatase, snake venon phosphodiesterase, and T2 RNase was systematically checked.