A Raman microscope and Raman difference spectroscopy are used to detect the vibrational signature of RNA-bound magnesium hydrate in crystals of hepatitis delta virus (HDV) ribozyme and to follow the effects of magnesium hydrate binding to the nonbridging phosphate oxygens in the phosphodiester backbone. There is a correlation between the Raman intensity of the innersphere magnesium hydrate signature peak, near 322 cm(-1), and the intensity of the PO2- symmetric stretch, near 1100 cm(-1), perturbed by magnesium binding, demonstrating direct observation of -PO2-center dot center dot center dot Mg2+(H2O)(x) innersphere complexes. The complexes may be pentahydrates (x = 5) and tetrahydrates (x = 4). The assignment of the Raman feature near 322 cm-1 to a magnesium hydrate species is confirmed by isotope shifts observed in D2O and (H2O)-O-18 that are semiquantitatively reproduced by calculations. The standardized intensity changes in the 1100 cm(-1) PO2- feature seen upon magnesium hydrate binding indicates that there are similar to 5 innersphere Mgt(2+)center dot center dot center dot -O2P contacts per HDV molecule when the crystal is exposed to a solution containing 20 mM magnesium.