Capillary electrophoresis has been used to characterize the interaction of monovalent cations with 26-basepair DNA oligomers containing A-tracts embedded in flanking sequences with different basepair compositions. A 26-basepair random-sequence oligomer was used as the reference; lithium and tetrabutylammonium (TBA+) ions were used as the probe ions. The free solution mobilities of the A-tract and random-sequence oligomers were identical in solutions containing <∼100 mM cation. At higher cation concentrations, the A-tract oligomers migrated faster than the reference oligomer in TBA+ and slower than the reference in Li+. Hence, cations of different sizes can interact very differently with DNA A-tracts. The increased mobilities observed in TBA+ suggest that the large hydrophobic TBA+ ions are preferentially excluded from the vicinity of the A-tract minor groove, increasing the effective net charge of the A-tract oligomers and increasing the mobility. By contrast, Li+ ions decrease the mobility of A-tract oligomers because of the preferential localization of Li+ ions in the narrow A-tract minor groove. Embedding the A-tracts in AT-rich flanking sequences markedly alters preferential interactions of monovalent cations with the B∗ conformation. Hence, A-tracts embedded in genomic DNA may or may not interact preferentially with monovalent cations, depending on the relative number of A·T basepairs in the flanking sequences.