Competitive binding of the most common cations of the cytoplasm (K(+), Na(+), Ca(2+), and Mg(2+)) with DNA was studied by equilibrating oriented DNA fibers with ethanol/water solutions (65 and 52% v/v EtOH) containing different combinations and concentrations of the counterions. The affinity of DNA for the cations decreases in the order Ca > Mg >> Na approximately K. The degree of Ca(2+) and/or Mg(2+) binding to DNA displays maximum changes just at physiological concentrations of salts (60-200 mM) and does not depend significantly on the ethanol concentration or on the kind of univalent cation (Na(+) or K(+)). Ca(2+) is more tightly bound to DNA and is replaced by the monovalent cations to a lesser extent than is Mg(2+). Similarly, Ca(2+) is a better competitor for binding to DNA than Mg(2+): the ion exchange equilibrium constant for a 1:1 mixture of Ca(2+) and Mg(2+) ions, K(c)(Ca)(Mg), changes from K(c)(Ca)(Mg) approximately 2 in 65% EtOH (in 3-30 mM NaCl and/or KCl) to K(c)(Ca)(Mg) approximately 1.2-1.4 in 52% EtOH (in 300 mM NaCl and/or KCl). DNA does not exhibit selectivity for Na(+) or K(+) in ethanol/water solutions either in the absence or in the presence of Ca(2+) and/or Mg(2+). The ion exchange experimental data are compared with results of grand canonical Monte Carlo (GCMC) simulations of systems of parallel and hexagonally ordered, uniformly and discretely charged polyions with the density and spatial distribution of the charged groups modeling B DNA. A quantitative agreement with experimental data on divalent-monovalent competition has been obtained for discretely charged models of the DNA polyion (for the uniformly charged cylinder model, coincidence with experiment is qualitative). The GCMC method gives also a qualitative description of experimental results for DNA binding competitions of counterions of the same charge (Ca(2+) with Mg(2+) or K(+) with Na(+)).