Abstract A previous report (Bouché, J.P.C. (1973) J. Bacteriol. 115, 752–761) has presented evidence that a part of the DNA synthesized in a methionine starved dnaB mutant after return to the permissive temperature is completely or almost completely unmethylated. We describe here the isolation of this DNA, and either its methylation in vitro by Escherichia coli K methylases, or its restriction by endonucleases K and RI from E. coli, and dII from Haemo-philus influenzae. These experiments show that this DNA not only lacks methylation due to the general (non-modifying) methylases, but also due to the K modification methylase. Furthermore, the levels of restriction of this DNA were found to be equivalent to those of an E. coli K DNA deficient only in modification. We conclude that there is no significant overlap in vitro between the sequence specificities of major non-modifying methylases and of the endonucleases tested.