The substrate specificity of the interferon-induced mouse L-cell enzyme, 2',5'-oligoadenylate synthetase, was determined with a number of nucleoside 5'-triphosphate analogues. Selected nucleoside 5'-triphosphates were converted to 2',5'-oligonucleotides with the following order of efficiency for the nucleoside: 8-azaadenosine greater than adenosine = 2-chloroadenosine greater than sangivamycin greater than toyocamycin greater than formycin greater than 3-ribosyladenine greater than ribavirin greater than tubercidin greater than adenosine 1-oxide greater than 2-beta-D-ribofuranosylthiazole-4-carboxamide greater than inosine = 1,N6-ethenoadenosine greater than guanosine greater than 8-bromoadenosine = uridine greater than cytidine. Adenosine 5'-((beta, gamma-imidotriphosphate) did not seem to be a recognizable substrate since no detectable product resulted. Either the 2',5'-oligoadenylate synthetase is not as specific as had been previously thought, or there may be more than one 2',5'-oligonucleotide synthetase. The 2',5'-oligonucleotide analogue products in which the adenosine of ppp(A2'P5')nA was replaced by the various nucleoside analogues were separated by DEAE-cellulose column chromatography and the chain length and number of 5'-phosphate residues analyzed by a rapid, efficient high-performance liquid chromatographic (HPLC) system involving ion-pairing C18 reversed-phase column chromatography. Separation of the 5'-mono-, 5'-di-, and 5'-triphosphorylated forms of the 2',5'-oligonucleotide analogue dimers, trimers, tetramers, and pentamers was readily achieved by this useful HPLC system. No 5'-nonphosphorylated forms were detected for any of the 2',5'-oligonucleotide analogue products.