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This article describes the results of a multi-analytical research carried out on the mural paintings from Évora Cathedral, one of the oldest and most prominent monuments of Évora city. The paintings were a late 16th Century addition to the cathedral's northern transept chapel and are attributed to the painter José de Escovar, whose painting workshop was remarkably active in the so-called golden age of mural paintings in southern Portugal. The aim of the study was to reconstruct the paintings' life story through a materials science perspective by discerning the material characteristics of the original and overpaint campaigns. The analytical setup comprised technical photography (TP) in the visible and UV light, Vis-Spectrophotometry, optical microscopy (OM) complemented with scanning electron microscopy with energy dispersive X-ray spectrometry (SEM-EDS), micro Fourier transform infrared spectrometry (µFT-IR) and pyrolysis-gas chromatography (Py-GC/MS) analyses of the collected samples. Results show that the original paint layers were executed with a combination of fresco and secco techniques, using a very simple chromatic palette composed of earth pigments, mercury sulphide, calcium carbonate and green copper based pigments. The paintings were heavily retouched and overpainted using earth pigments, smalt, Prussian blue, zinc white and lead white. Furthermore, the analyses of the cross-sections taken from the stuccoed frames revealed a first decorative campaign made with gold and silver alloy. © 2016 Wiley Periodicals, Inc. Col Res Appl, 41, 276-282, 2016 [ABSTRACT FROM AUTHOR]