Palaeolithic and modern reindeer antler were analysed using complementary techniques to evaluate the conservation state of bone material. X-ray diffraction, FT-IR spectroscopy, scanning electron microscopy (SEM–EDX) and transmission electron microscopy (TEM–EDX) as well as proton induced X-ray and γ-ray emission (PIXE–PIGE) at the particle accelerator AGLAE, C2RMF were applied. This study enabled us to define the chemical and structural features of ancient antler at the micro- and nanometre scale and compare them to those of other bone materials such as bone and ivory. Antler is richer in its organic fraction compared to bone and ivory. Its mineral fraction, carbonated hydroxylapatite, shows very specific crystal shapes at the nanometre scale. This specific property allowed us to recognize antler traces in pigment samples originating from rock art paintings in the Lascaux cave, Dordogne, southwestern France, Magdalenian period. Therefore, TEM–EDX is the technique of choice for characterizing antler traces in complex mixtures. In the paint material of Lascaux, antler seems most likely to be a pollutant introduced either by stirring the pigments in water with a piece of antler or by carving antler artefacts next to the preparation of paint material. However, it could eventually be used as a marker of paintings that were created contemporaneously.