Conversion of a centrally located phosphate group to an electrically neutral methyl phosphonate in a four-way DNA junction can exert a major influence on its conformation. However, the effect is strongly dependent on stereochemistry. Substitution of the proR oxygen atom by methyl leads to conformational transition to the stacking conformer that places this phosphate at the point of strand exchange. By contrast, corresponding modification of the proS oxygen destabilizes this conformation of the junction. Single-molecule analysis shows that both molecules are in a dynamic equilibrium between alternative stacking conformers, but the configuration of the methyl phosphonate determines the bias of the conformational equilibrium. It is likely that the stereochemical environment of the methyl group affects the interaction with metal ions in the center of the junction.