The physical properties of nucleic acid helices strongly depend on their interaction with counterions and water. Of special interest is the structure of their ionic atmosphere which is closely related to its overall hydration because the distances between counterions and the atomic groups of a helix are comparable to the size of a water molecule. In an effort to answer whether the putative higher linear charge density of triple helices, relative to duplexes, exerts special effects on the structural and hydration parameters of their ionic atmosphere, we used a combination of high-precision ultrasonic velocity and density techniques to follow the hydration effects upon substituting Na+ for Cs+ or Mg+ ions in the ionic atmosphere of poly(rA). 2poly(rU) and poly(rA) poly(rU). The titration of the Na+ salt of each helix with Cs+ results in marginal hydration effects. This indicates that both Na+ and Cs+ form outer-sphere counterion-RNA complexes, where the counterion keeps its coordinated water molecules. The substitution of Na+ for Mg2+ results in positive compressibility values (3 x 10(-4) cm(3)/bar per mol of nucleotide) and volume effects (2 cm(3) per mol of nucleotide), and reflects a small dehydration of the whole Mg2+-RNA complexes. The overall dehydration level corresponds to the formation of outer-sphere complexes, indicating that Mg2+ keeps most of its coordinated water in the ionic atmosphere of each helical structure. The resulting small effects in the hydration parameters of the tripler ionic atmosphere suggest that the structure of the counterion-triplex complex may be determined by short-range interactions, including the immobilization of water molecules, while the long-range electrostatic interactions that determine the condensation of counterions do not show a significant influence on the local structure of the ionic atmosphere of these helices.