The drug, 9-,6-D-arabinofuranosyl adenine, selectively inhibits the synthesis of nuclear DNA without affecting extrachromosomal DNA synthesis in rat cells transformed by Rous sarcoma virus (B-mix K-44/6 To explore the role of extrachromosomal DNA in virustransformed cells, we have begun to examine circular DNA from a line of mammalian cells transformed by Rous sarcoma virus (RSV). B-mix K44/6 cells were subcloned from a population of embryonic rat cells transformed in vitro by the Prague strain of RSV (1). These large epithelioid cells are malignant for homologous rat hosts and have been shown by both in vivo experiments (1) and cell fusion studies with chick-embryo fibroblasts (Shipman, unpublished observations) to contain the genome of RSV. Circular DNA represents a very small fraction of the total DNA of a mammalian cell. Therefore, to aid in the isolation of circular DNA it would be desirable to inhibit nuclear DNA synthesis without affecting the replication of circular DNA. A selective inhibition of this nature has been accomplished in prokaryotic cells. Helinski and Clewell (2) have described a preparation using chloramphenicol, where Col E1 plasmid DNA synthesis occurs in the absence of chromosomal DNA synthesis. Similarly, in synchronous cultures of Chlorella, synthesis of nuclear DNA was inhibited by more than 90% in the presence of 15 ,pM cycloheximide, whereas the inhibitor had little effect on satellite DNA synthesis (3). Although the synthesis of cytoplasmic DNA in HeLa cells is somewhat resistant to inhibition by high concentrations of hydroxyurea (4), the selective inhibition of nuclear DNA synthesis has not been described in mammalian cells.Abbreviations: ara-A, 9-0-D-arabinofuranosyl adenine; ara-ATP, the 5'-triphosphate of ara-A; Col El, colicinogenic factor E1; HBS, HEPES-buffered saline (see ref. 12); HEPES, 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid; PI, propidium diiodide; RSV, Rous sarcoma virus; SDS, sodium dodecyl sulfate.