Previously we reported on the presence of a high (2'-5')oligoadenylate synthetase activity in the marine sponge Geodia cydonium [Kuusksalu, A., Pihlak, A., Müller, W. E. G. & Kelve, M. (1995) Eur. J. Biochem. 232, 351-357]. The presence of (2'-5')oligoadenylates [(2'-5')A] in crude sponge extract was shown by radioimmunoassay and by their HPLC comigration with authentic (2'-5')A oligomers. In addition, the sponge (2'-5')oligoadenylates displayed biological activity, as determined by inhibition studies of protein biosynthesis in rabbit reticulocyte lysate. In the present study individual (2'-5')oligoadenylates synthesized by sponge enzyme were separated by HPLC. The exact composition of every oligonucleotide peak eluted was determined by matrix-assisted laser-desorption-ionization mass spectrometry (MALDI-MS) analysis. The 2'-5' phosphodiester bond in oligoadenylates was verified by NMR analysis. Based on the high concentration of (2'-5')A oligomers in G. cydonium and their similarity with those found in mammals we propose that the (2'-5')A system is involved in a cytokine-mediated pathway and/or in a protection system against viruses, present in the marine environment.