Heteroduplex formation between imperfectly homologous DNA sequences may result in the formation of a four-way junction at which non-Watson-Crick base mismatches are present at the point of strand exchange. This raises the question of the effect of such mismatches on the structure and stability of these potential recombination intermediates. We have constructed a series of four-way DNA junctions containing single-base mismatches, and have studied the structure of the junctions by means of gel electrophoresis and chemical modification. We observed a range of effects on the structure of the junction, ranging from almost total abolition of folding through to normal accommodation into the folded structure. In some cases we observed gel electrophoretic data consistent with a dynamic equilibrium between folded and unfolded conformations, and in general the folded form was favoured at higher concentrations of cation. The effects of single mismatches on the structure of the four-way junction may be summarized in terms of: (1) the nature of the mismatch, where we note a correlation between the thermal stability of a given mismatch and its ability to be accommodated into a folded junction; or (2) the sequence context, where the effect of a given mismatch on the structure of a junction depends on the neighbouring base-pairs. These factors are illustrated by a junction, containing a C.A mismatch, that adopted alternate isomeric conformations dependent upon pH; as the state of protonation of the mispair changed, the structure was altered along with the interaction with neighbouring base-pairs. Most base mismatches may be accommodated into the folded stacked X-conformation of the four-way junction, but many require elevated cation concentration to permit the folding process to proceed. Some mismatches were found to be extremely destabilizing.