Dimethyl sulfoxide (DMSO) is commonly used as a cosolvent to improve the aqueous solubility of small organic compounds. Its use in a screen to identify novel inhibitors of the enzyme NAD+ synthetase led to this investigation of its potential effects on the structure and stability of this 60-kD homodimeric enzyme. Although no effects are observed on the enzyme's catalytic activity, as low as 2.5% (v/v) DMSO led to demonstrable changes in the stability of the dimer and its unfolding mechanism. In the absence of DMSO, the dimer behaves hydrodynamically as a single ideal species, as determined by equilibrium analytical ultracentrifugation, and thermally unfolds according to a two-state dissociative mechanism, based on analysis by differential scanning calorimetry (DSC). In the presence of 2.5% (v/v) DMSO, an equilibrium between the dimer and monomer is now detectable with a measured dimer association constant, Ka, equal to 5.6 × 106/M. DSC curve analysis is consistent with this finding. The data are best fit to a three-state sequential unfolding mechanism, most likely representing folded dimer ⇄ folded monomer ⇄ unfolded monomer. The unusually large change in the relative stabilities of dimer and monomer, e.g., the association equilibrium shifts from an infinitely large Ka down to ∼10 6/M, in the presence of relatively low cosolvent concentration is surprising in view of the significant buried surface area at the dimer interface, roughly 20% of the surface area of each monomer is buried. A hypothetical structural mechanism to explain this effect is presented.