Paintings often consist of highly complex layered structures that contain a mixture of organic and inorganic materials at each layer depending upon the artist’s technique. A comprehensive analysis of the chemical composition could provide critical information on the sequence of events that led to an artwork’s current state. In this investigation, we have employed a novel imaging technique known as Secondary Ion Mass Spectrometry (SIMS) to chemically map the cross-section of a paint film sampled from Leonardo da Vinci’s Adoration of the Magi at a submicron resolution using a C60+ ion source. A wide range of materials were found to be spatially localized at the different layers such as: protein, carbon, silicates, fatty acids, salts and lead associated compounds. An interaction of chloride salts with the lead white was observed in the priming layer. The chloride salts disrupted most of the priming layer suggesting that a cleaning process took place that removed most of the priming layer before the monochrome brown repaints were added on top at a later time.