This paper employs various techniques to analyze the mixture of chalk and binder materials used, by Portuguese and Flemish painters in the 15th and 16th centuries, to enhance the reflection of light in paintings. The cases studied show evidence of the search by painters for light effects created when combining specific fillers and binders to obtain absorbent or non-absorbent ground layers in order to reflect superficial or deep light in paintings. These brightening effects are largely provided by microscopic coccoliths and calcispheres-the main constituents of chalk. The composition, size and slightly concave-convex shield-like shape of calcareous nannofossils (micrometrical dimensions) also facilitate application, thereby increasing the speed of handling. These calcareous nannofossils are crucial proof that chalk was used in the ground layers of Portuguese paintings. They have proved to be important in defining the various stages of Portuguese painting workshops, such as the Viseu Workshop (1501-1569), which used powdered chalk in the first phase and powdered limestone in the second phase in the production of a ground layer. A two-layer structure has been verified in some Flemish paintings of the period, but the use of different binders to provide different levels of light absorption and reflection in these artworks had not been previously identified. The results showing this two-layer ground structure are significant in making the connection between Flemish and Portuguese art in the context of a specific painting technique. The use of calcium carbonate ground layers was verified by SEM-EDS and confirmed by μ-XRD, and μ-Raman, while binders were analyzed by μ-FTIR and optical microscopy, using staining tests.