The exact physiological function of Ap 3 A (A5'ppp5''A, 5'5'' diadenosine triphosphate) remains unclear. Previously we have demonstrated that the human p46 2-5A synthetase (OAS1) efficiently utilises Ap 3 A as an acceptor substrate for oligoadenylate synthesis. Here we show that Ap 3 A(2'p5'A) n oligonucleotides can activate the 2-5A-dependent RNase (RNase L), when the number of 2',5'-linked adenyl residues is two or more. Under the experimental conditions applied the half-maximal activation (AC 5 0 ) of RNase L for 2'-adenylated Ap 3 A derivatives was determined to be in nanomolar range while the AC 5 0 for 2-5A 3 was 0.4 nM. The Ap 3 A(2'p5'A) n oligonucleotides are thus less effective in activating RNase L than 2-5A. We also investigated the occurrence of 2'-adenylated Ap 3 A in interferon and poly(I).poly(C)-treated HeLa cells. In purified trichloroacetic acid-soluble extracts about 40% of RNase L-activating material is resistant to phosphatase treatment, whereas the removal of 5'-terminal phosphates greatly reduces the activating properties of 2-5A. We assume that this activity at least partly may be associated with the presence of 2'-adenylated Ap n A derivatives with blocked 5'-terminal phosphates.