Single crystals of pure diopside (CaMgSi2O6) were synthesized at 6 GPa or 8 GPa and between 1300 °C and 1150 °C under water-saturated conditions coexisting with forsterite + enstatite, enstatite + coesite, wollastonite + coesite or forsterite + wollastonite, and the incorporation mechanisms of OH in the pyroxenes in the respective phase assemblage were studied by FTIR-spectroscopy. The IR spectra of diopside exhibit two different OH-absorption bands: one prominent band at 3357 cm−1 that appears in all spectra, whereas another band at 3598 cm−1 only appears in spectra of diopside synthesized under lower silica activity (i.e., coexisting with forsterite). The band at 3598 cm−1 indicates a low availability of Si and can be assigned to tetrahedral OH-defects (T-site vacancies), while the band at 3357 cm−1 can be assigned to octahedral defects (M-site vacancies). Enstatite crystals from the assemblages diopside + enstatite + coesite and diopside + enstatite + forsterite exhibit an additional absorption band at 3436 cm−1 when compared to enstatite synthesized in the system MgO–SiO2–H2O. This absorption band could be caused by generation of additional hydrous defects due to the incorporation of Ca that amounts up to 1.8 wt% CaO in enstatite coexisting with diopside. The water content of diopside coexisting with enstatite and coesite clusters around 145 ppm (wt), while the water content of diopside from all other phase assemblages clusters around 60 ppm (wt). The new results suggest that the water content in pyroxenes correlates with silica activity during formation.