DNAs of adenovirus type 2 and type 12 contain low amounts of methylated bases (0.01 and 0.02% N6-methyladenine per adenine, if any, and 0.04 and 0.06% 5-methylcytosine per cytosine for type 2 and type 12, respectively), whereas the DNA of the mammalian host cells contains much more 5-methylcytosine (3.57% for human KB cells). The Methylation of DNA was determined by a sensitive method based on two consecutive steps of two-dimensional thin-layer chromatography of the radioactively labeled DNA bases. By this procedure the detection limits of 5-methylcytosine and N6-methyladenine could be lowered to 0.01% per main base. During replication of adenoviruses in permissive human cells, a large amount of viral DNA is synthesized in the nucleus of the host cell (1-3). Viral DNA accounts for 30-50% of the total intracellular DNA at late times after infection (2, 3). Concomitantly with the onset of viral DNA replication, host DNA synthesis is drastically reduced (4).In previous DNA base analyses of mammalian cells 3-6% 5-methylcytosine (MeCyt) per cytosine (Cyt) was found (5, 6), while N6-methyladenine (MeAde) could not be detected (7,8).Little is known about the biological role of DNA methylation. There are reports on tissue specificities in the content of MeCyt in mammalian cells (5,6,9). Certain developmental stages are associated with variations in the content of methylated DNA bases in pro-and eukaryotes (10, 11). The only established function of certain DNA methyltransferases is the modification of DNA in bacteria leading to resistance against restriction endonucleases of the same specificity (12, 13).Since it is presumed that synthesis of adenovirus DNA in human cells is mediated, at least in part, by the host replication system, one would expect viral DNA to show the methylation pattern characteristic of host DNA. However, in contrast to host -DNA, adenovirus DNA has a low level of methylation. Comparison of untransformed cells and cells transformed by adenovirus has shown a difference in the levels of DNA methyl- (Calbiochem). The method of further purification has been described (21).In some experiments the total cellular DNA and the nonencapsidated viral DNA were isolated from Ad2-infected KB cells 40-48 hr after infection. Cell extracts were prepared by ultrasonic treatment, and the virions were isolated by equilibrium sedimentation in CsCl density gradients with a mean density of 1.334 g/cm3 as described (3). All free intracellular DNA was recovered from the pellet of this gradient and recentrifuged to equilibrium in a CsCl density gradient with a mean density of 1.70 g/cm3.Purification of Viral and Cellular DNA. The DNA was precipitated with two volumes of ethanol and kept for 4 hr at -20°. Traces of RNA were removed by hydrolysis in 0.25 M KOH 16-18 hr at 37°. Subsequently, the DNA was again pre-3923