Single crystals of synthetic CaMgSi2O6 (diopside) doped with different amounts of Fe and Na were produced under water- and silica-saturated conditions at 20 kbar using a piston-cylinder apparatus. All samples were investigated by FTIR spectroscopy and electron microprobe, and some crystals were characterised by Mößbauer spectroscopy. IR spectra recorded on pure diopside show one OH absorption band at 3360 cm−1. In Na-doped diopside an additional band centred at 3428 cm−1 is observed. Crystals containing Na as well as Fe exhibit either an additional prominent peak at 3428 cm−1 or one at 3443 cm−1 except for one sample showing also a band at 3651 cm−1. The 1:1 dependency between incorporated Na and H in samples doped with only Na suggests that all sodium is coupled to hydrogen in the range of Na-content investigated in this study. In contrast, crystals doped with Fe and Na contain very different amounts of hydrogen, which is interpreted to be caused by several interacting mechanisms: in one mechanism the hydrous defect is replaced by an anhydrous aegirine compound, in another one the aegirine compound is replaced by the combination of a hydrous Fe-associated defect and an anhydrous Na-associated defect. The Fe-related hydrous defects could also be destroyed by a third mechanism, which allows the incorporation of ferric iron without being coupled to monovalent cations. The band at 3428 cm−1 can be assigned to a Na-related OH-defect. Bands at 3360 and 3443 cm−1 showing very similar behaviour may both be related to M-site vacancies, and the band at 3443 cm−1 seems to be caused by replacement of two coordinating Mg by a ferric iron and a proton. For the 3651 cm−1 peak no distinct assignment can be proposed. The results of this study show that different hydrous defects may transform into anhydrous defects, reducing hydrogen solubility in the crystal structure. Thus, water content in clinopyroxene is not a simple function of the total amount of mono- and trivalent cations. In natural clinopyroxene crystals from upper-mantle peridotites, which are Na- and Fe-bearing, neither the Na-associated band at 3428 cm−1 nor the Fe-associated band at 3443 cm−1 is observed. This fact suggests that other mechanisms, e.g. interactions between hydrous defects and other cations, most probably Al, could have influence on OH incorporation in natural clinopyroxenes.