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The dark colour of some excavated architectural elements of the Ploutonion, in the archaeological site of Hierapolis of Phrygiae (Turkey), was investigated. The Ploutonion, visited by numerous ancient writers, such as Cicero and Strabo, was a sanctuary dedicated to Pluto and Kore, constructed during the early Imperial period above the entrance to a natural cave, from which thermal waters and poisonous gases emerge. It was used for sacred rituals: sacrifices, incubation and healing. A multi‐analytical approach (X‐ray diffraction, X‐ray fluorescence, optical and electronic microscopy, and chemical analyses of the thermal spring water) was used in order to define the composition and origin of darkening phenomena of some architectural elements of the Ploutonion. [ABSTRACT FROM AUTHOR]