The free solution mobilities of curved and normal 199-bp DNA fragments have been measured in buffer solutions containing various quantities of the organic cosolvents methanol, ethanol, 2-propanol, 2-methyl-2,4-pentanediol (MPD), ethylene glycol, and ACN, using CE. The curved fragment, taken from the VP1 gene of SV40, contains five unevenly spaced A- and T-tracts in a centrally located "curvature module"; the A- and T-tracts have been mutated to other sequences in the normal 199-bp fragment. The free solution mobility of the curved 199-bp fragment is significantly lower than that of its normal counterpart in aqueous solutions [Stellwagen, E., Lu, Y. J., Stellwagen, N. C., Nucleic Acids Res. 2005, 33, 4425-4432]. The mobilities of both the curved and normal fragments decrease with increasing cosolvent concentration, due to the effect of the cosolvent on the viscosity and dielectric constant of the solution. The mobility differences between the curved and normal 199-bp fragments and the mobility ratios decrease approximately linearly with the increasing mole fraction of cosolvent in the solution. Hence, MPD and other organic cosolvents affect DNA electrophoretic mobility by a common mechanism, most likely the preferential hydration of the DNA surface that occurs in aqueous cosolvents. The gradual loss of the anomalously slow mobility of the curved 199-bp fragment with increasing cosolvent concentration, combined with other data in the literature, suggests that preferential hydration gradually widens the narrow A-tract minor groove, releasing site-bound counterions in the minor groove and shifting the conformation toward that of normal DNA.