Long DNA palindromes present a threat to genomic stability and are not tolerated in Escherichia coli. It has been suggested that this is a consequence of cruciform or hairpin formation by palindromic sequences. This work describes a methylation inhibition assay for unusual DNA secondary structure in vivo that is both internally controlled and non-invasive. If a palindrome with a central GATC target site for Dam methylase assumes a cruciform or hairpin conformation in vivo, then the GATC sequence will be located in a single-stranded loop and will consequently not be modified. The centre of a long perfect palindrome located in bacteriophage lambda is shown to be methylation-resistant in vivo. Changes to the central sequence and insertions of 10 base-pairs of asymmetric sequence do not alter the degree of under-methylation, but insertions of 20 base-pairs or more of asymmetric sequence reduce the under-methylation of the palindrome centre. We also show that the centres of long palindromes are more under-methylated than equivalent sequences in a non-palindromic context. These results are consistent with an unusual secondary structure, such as DNA cruciform or hairpin, and indicate that the formation pathway of the structure detected is independent of the composition and symmetry of the central 10 base-pairs of the palindrome.