A density and radioisotopic labeling procedure has been used to study the in vivo pattern of DNA methylation in Escherichia coli. The DNA of an auxotrophic strain of E. coli (thymine-, methionine-, arginine, and tryptophan-), starved of arginine and tryptophan but not methionine, did not undergo additional methylation during subsequent cell growth in the presence of methyl-labeled methionine. However, the DNA of methionine-starved cells was subsequently methylated upon the addition of methionine. When methyl-labeled methionine was added to exponentially growing bacteria, only that portion of the DNA then being synthesized was methylated. Neither the unreplicated parental DNA nor the replicated parental template strand was methylated. It is concluded that DNA methylation normally occurs in the nascent DNA strand close to or at the replication point. © 1968.