The opening of base pairs of double-stranded DNA is an important process, being a prerequisite for replication and transcription and possibly a factor in the recognition, flexibility and structure of DNA. The kinetics of base-pair opening have, however, been controversial. Base-pair opening can be studied by following the exchange of protons from imino groups with water, a process that seems only to occur from open base pairs. We have recently demonstrated catalysis by proton acceptors of imino proton exchange in nucleic acids. This has enabled us to determine the base-pair lifetimes, which are in the region of 10 ms at room temperature1,2. In earlier reports it had been considered that proton exchange is limited by the rate of base-pair opening, which had led to estimates of base-pair lifetimes that were larger by one or two orders of magnitude3–17. There are also important discrepan-cies between recent and early estimates of the base-pair dissociation constant. Earlier estimates of base-pair lifetimes correspond in fact to the time required for proton exchange in the absence of added catalyst (AAC exchange). This could be a distinct mode of base-pair opening with a very long open lifetime, different from the mode revealed by the effect of catalyst. The evidence reported here suggests on the contrary that there is only a single mode of base-pair opening and that proton exchange in the absence of added catalyst is in fact catalysed by a proton acceptor intrinsic to the nucleic acid, most probably the other base of the open pair.