The minor groove hydration spine is a key feature of the crystal structure of the B-DNA dodecamer duplex [d(CGCGAATTCGCG)](2). At the floor of the groove, water molecules bridge bases from opposite strands by hydrogen bonding to N3 and O2 atoms of adenine and thymine, respectively. However, the interpretation that the series of electron density peaks lining the groove represents indeed water molecules, while generally agreed upon, remains an assumption. The limited resolutions of dodecamer crystal structures have thus far made it impossible to reliably distinguish between water and monovalent metal cations, such as Na+, normally present in the crystallization buffer. Using X-ray diffraction data to near-atomic resolution of dodecamer crystals grown in the presence of either Rb+ or Cs+ cacodylate, we have tested the possibility of alkali metal ion coordination in the minor groove. The structural data are consistent with a single Rb+ intruding the hydration spine at the central ApT step. The ion has partial occupancy and replaces the water molecule that links the keto oxygens of thymines from opposite strands. The observed dimensions of the binding site suggest preferred binding of Rb+ or K+, while Na+ or Cs+ may be prevented from binding stably. Therefore, minor groove ion coordination appears to be an isolated event, highly sequence dependent and unlikely to significantly affect the particular geometry of the A-tract in the Dickerson-Drew dodecamer. In addition to allowing a distinction between water and alkali metal ions, the high-resolution crystal structures provide a more complete picture of the minor groove water structure: four fused water hexagons dissect the central portion of the minor groove, with the inner corners of the hexagons coinciding with the original spine water positions. Thus, it may be more appropriate to refer to this arrangement as a ribbon of hydration instead of a spine of hydration.