Extensive PIXE investigations have been performed at the LABEC accelerator laboratory in Florence on the painting "Ritratto Trivulzio" by Antonello, da Messina, one of the great Italian masters of XV Century and a pioneer in modern oil painting. It is well known that a complete and unambiguous characterisation of materials in paintings is often difficult, owing to their complex structure. For the "Ritratto Trivulzio", the combination of advanced variants of PIXE, such as differential and scanning-mode analysis, provided a relevant contribution to the characterisation of paint layers, in terms of composition and structure, in a totally non-invasive and non-destructive way. Single-spot mode PIXE with external proton beams of about half millimetre diameter was first used for a general characterisation of different areas of the painting. Differential PIXE performed in many spots led to reconstruct the sequence of paints employed by the artist and to estimate in some cases the local paint layer thickness. A second run carried out with the external scanning microbeam facility, using a 3 MeV proton beam of about 80 mu m size, was then crucial to clarify some issues raised by the first analysis. In particular, elemental maps from selected areas helped to understand the way some colour shades had been obtained on the red mantle of the portrayed gentleman, using different pigments irregularly distributed on the surface. (C) 2009 Elsevier B.V. All rights reserved.