Sequence-dependent coordination of alkali ions to the nucleotide bases in the minor groove of AT-tract B-DNA has recently been inferred from X-ray crystallography, solution NMR and computer simulations. Here, we present new Na-23 and Rb-87 magnetic relaxation dispersion (MRD) data that demonstrate competitive and long-lived binding of Na+ and Rb+ ions in the minor groove of the B-DNA duplex [d(CGCGAATTCGCG)](2). The Na+/Rb+ selectivity of the minor groove is found to be weak, consistent with local structural flexibility. The ion occupancies derived from the MRD data are substantially higher than previously reported, suggesting that groove-bound ions significantly influence the energetics and structural polymorphism of DNA in vivo. For example, in the presence of 0.20 M Na+ and 0.56 M Rb+ at 4 degreesC, the ApT site in the minor groove is occupied by a Rb+ ion, a Na+ ion, or a water molecule 40, 10, and 50% of the time, respectively. In the absence of Rb+, the Na+ occupancy increases to 50%. At 4 degreesC, the mean residence time of groove-bound ions is 0.2 +/- 0.1 mus for Rb+ and 10 ns to 100 mus for Na+. A shorter correlation time of 2 ns is attributed to counterions bridging cross-strand phosphate groups.