Competitive binding of K+, Na+, and Li+ to DNA was studied by equilibrating oriented DNA fibers with ethanol/water solutions in the range of ethanol concentration from 65 to 90% (by volume) and for salt concentrations, C-s, from 3 to 300 mM. The affinity of DNA for the cations decreases in the order Na approximate to K > Li, and this is opposite to the sequence determined for DNA in aqueous solution. The ion exchange equilibrium constant, K-c(Li)K, determined in the system DNA fibers-ethanol/water solutions of KCl and LiCl, varies between K-c(Li)K approximate to 1.4 in 70% EtOH (K/Li = 1/1) and K-c(Li)K approximate to 2.5-2.7 in 84-90% EtOH (K/Li = 1/1) or K-c(Li)K approximate to 3.7-4.0 in 84% EtOH (K/Li = 1/9), Between 76 and 84% EtOH, the value of K-c(Li)K increases steeply, which is due to the B-A transition of KDNA occurring in this concentration range of EtOH. Neither the A nor the B form of DNA exhibits selectivity for Na+ or K+ in mixtures of KCI and NaCl in ethanol/water solutions. Computer simulations based on the grand canonical Monte Carlo (GCMC) method were applied for modeling the experimental conditions. These calculations were performed within the approximations of describing the solvent as a dielectric continuum and the DNA polyion as a uniformly charged cylinder or a cylinder with arrays of spherical charges representing phosphate groups of the B or A form of DNA. It is found that the GCMC method explains qualitatively the ion selectivity of DNA in K/Li mixtures with respect to the dependence on the ethanol concentration, K/Li ratio, and A or B structural form of DNA.