The disorganization of the aqueous helical configuration of deoxyribonucleic acid (DNA) by a number of structurally related organic solvents has been investigated. In all the solvents investigated, denaturation is accompanied by 35–50% increase in absorbance (at 259 mμ) and a decrease in optical rotation (at 436 mμ) of 200–350 °. The following observations have been made: (a) The effectiveness of the denaturant increases with both chain length and increasing hydrocarbon content. Thus ethyl and propyl alcohols were found to be more effective than methanol. (b) The alkyl-substituted solvents N,N′-dimethylformamide, dimethyl sulfoxide, and tetramethylurea are the most effective denaturants among the various solvents employed. The midpoints of the denaturation transition due to these solvents in the presence of 1–5 × 10−2M salt, range from 19 to 27 mole % (57–62 vol.%), and the changes produced in optical rotation (at 436 mμ) upon denaturation are of the order of −300 to −350 °. (c) N,N′-dimethylformamide is a more effective DNA denaturant than formamide. (d) Increasing the hydroxyl content of the solvent, on the other hand, had no significant effect; the denaturation midpoints in methanol-water and ethylene glycol-water mixtures (in the presence of 0.5–5 × 10−2M salt) occur at 80 ± 1 mole % (90 ± 2 vol.%) of the nonaqueous component. These observations demonstrate the importance of hydrophobic forces and argue against the assignment of the stability of the aqueous configuration of DNA solely to hydrogen bonds.