In the presence of the adenosine deaminase inhibitor erythro-9-[3(2-hydroxynonyl)]adenine microM concentrations of 2'-deoxyadenosine (dAdo) are toxic to nondividing human lymphoid cells and induce G1-phase arrest in T-leukemic lymphoblasts, effects which appear to be independent of ribonucleotide reductase inhibition by accumulated 2'-deoxyadenosine 5'-triphosphate. We sought to determine if 2'-deoxyadenosine 5'-triphosphate had effects similar to those of other cytotoxic adenosine analogues which are incorporated into polyadenylated RNA [poly(A)+ RNA]. In the presence of erythro-9-[3-(2-hydroxynonyl)]adenine, 8-14C]dAdo, at minimal cytostatic concentrations, was incorporated into the polyadenylate segments of cytoplasmic poly(A)+ RNA in the human T-leukemic lymphoblast line CCRF-CEM, and 70% of incorporated dAdo was in the 3'-terminal position. No DAdo was found in enzyme hydrolysates of nonpolyadenylated regions of poly(A)+ RNA or of poly(A)-RNA. Enzymic hydrolysis of polyadenylated segments from labeled poly(A)+ RNA yielded adenosine:dAdo ratios of approximately 55:1.