The solid state dehydration of inosine dihydrate has been investigated using powder X-ray diffraction, differential scanning calorimetry, thermal gravimetric analysis, dynamic vapor sorption, hot stage microscopy, 13C solid state NMR, and variable temperature Fourier transform infrared spectroscopy. The information obtained from these experiments has been combined with an analysis of the crystallographic packing of inosine dihydrate and its two anhydrous polymorphs to yield mechanistic insights into the modes of water loss. The results allow an understanding of why, surprisingly, the dehydration of the dihydrate is always found to give the anhydrous, metastable, alpha-form.