Alkylating agents such as N-ethyl-N-nitroso compounds produce G : C to A : T transition mutations in DNA owing to the ethylation of O⁶-guanine. It is assumed that these mutations occur on replication via mispairing of ethylguanine with thymine rather than the normal cytosine. Such a mispair could be either very stable compared with the normal G : C base pair or it could be less stable than a normal G : C base pair but cause little disruption to the DNA, so that it is not easily recognized by repair enzymes. In this study, the stability and conformation of a dodecamer containing O⁶-ethylguanine: thymine base pairs has been compared with that of a similar duplex containing a normal sequence. Using molecular dynamics to study a duplex DNA sequence containing O⁶-ethylguanine: thymine base pairs, we find a normal Watson–Crick alignment of the mispairs. However, this conformation is stabilised by only one or two hydrogen bonds depending on whether the conformation of the ethyl group is either the cis,trans or the trans,trans orientation with respect to the N(1) of guanine. Analysis of other conformational features and flexibility has shown that it is difficult to distinguish behaviour of the modified sequence with that of the normal sequence. Like the sequences containing O⁶-ethylguanine: cytosine, the modified O⁶-ethylguanine: thymine sequences are slightly less stable than those containing the normal Watson–Crick G : C sequence. However, both O⁶-cis,trans-ethylguanine: thymine and O⁶-trans,trans-ethylguanine: thymine cause very little disruption to the DNA helices in contrast to the behaviour of O⁶-ethylguanine: cytosine and thus the former modifications will be difficult to distinguish from normal sequences by repair enzymes.