3-Deazaadenosine, a known potent inhibitor of adenosylhomocysteine hydrolase, exerts an inhibitory effect on the reproduction of Rous sarcoma virus (RSV-BH) and on the malignant transformation of chick embryo cells by this virus. The inhibitory effect is reversible. The increased capacity for glucose uptake, a biochemical characteristic of chick embryo cells transformed by RSV-BH, is inhibited by 3-deazaadenosine, and the ability of the infected cells to grow in suspension is blocked. After prolonged exposure to 3-deazaadenosine, the morphological phenotype characteristic of transformed cells largely disappeared, and transformed cells resembled noninfected cells. Deazaadenosine inhibits the reproduction of Sindbis virus, Newcastle disease virus, and vesicular stomatitis virus to a lesser degree than RSV-BH. Deazaadenosine, 0.1 m M, has no effect on DNA or protein synthesis in cells, and only a slight effect on RNA synthesis. No incorporation of 3-deaza[ 14C]adenosine into cellular nucleic acids was found. Deazaadenosine produces an increase in the intracellular level of adenosylhomocysteine, with the concomitant appearance of a relatively large amount of 3-deazaadenosylhomocysteine; the ratio of intracellular adenosylmethionine to adenosylhomocysteine, or (adenosylhomocysteine + 3-deazaadenosylhomocysteine) is decreased from 150 to 19, and 1.4 respectively. It is postulated that 3-deazaadenosine inhibits virus activities by its ability to inhibit adenosylhomocysteine hydrolase, resulting in an inhibition of methylation reaction(s) required for virus growth and replication.