Nucleic acids were separated by ion-exchange chromatography on 30 x 4.6 and 100 x 4.6 mm columns packed with a micropellicular anion exchanger made of 3-microns rigid polystyrene-based non-porous microspheres with a covalently bound hydrophilic layer and DEAE functional groups at the surface. The stationary phase particles showed negligible swelling in methanol according to permeability measurements with water and methanol. Nucleic acids and their fragments including synthetic single-stranded oligonucleotides, linear, nicked and supercoiled DNAs as well as DNA restriction fragments were separated in less than 5 min, a time scale that is much smaller than that of conventional high-performance liquid chromatographic analysis for such samples. When only buffer and sodium chloride were used in the eluent for the separation of double-stranded DNA restriction fragments pGEM-3Z/Taq I, electrophoretic analysis of the effluent revealed the presence of smaller fragments in the bands of the larger ones. Upon addition of ethylenediaminetetraacetic (EDTA) salt to the eluent, however, such contamination by shorter fragments was no longer observed. In the absence of EDTA, magnesium chloride in the eluent at a concentration of 1 mM precluded the separation of the restriction fragments under otherwise identical chromatographic conditions.