Sex determination in fossil hominin remains is often challenging due to the fragmentary nature of the record and the limitations of morphological assessments. In this study, we apply a proteomic approach to assess the sex of an upper second molar (RV’23-350) from the Middle Pleistocene site of Ruidera (Spain). By analyzing the presence of amelogenin, a key enamel protein encoded by genes on both the X and Y chromosomes, we provide molecular evidence supporting the classification of this fossil as belonging to a male individual. To the best of our knowledge, this represents the first application of amelogenin-based sex estimation in European Middle Pleistocene hominin teeth. Our findings have significant implications for understanding hominin demographic structures and population dynamics in the Iberian Peninsula during this period. The ability to integrate proteomic analyses with traditional morphological approaches offers a novel and reliable methodology for paleoanthropological investigations, particularly in cases where skeletal preservation is insufficient for standard osteological assessments. This study highlights the potential of ancient protein analysis in refining taxonomic and biological reconstructions, further enhancing our knowledge of human evolution and variability in the Middle Pleistocene. • First application of amelogenin-based proteomic analysis for sex determination in a European Middle Pleistocene hominin tooth. • Molecular evidence suggests that the upper second molar RV’23-350 from Ruidera belonged to a male individual. • Proteomic methods enable reliable sex assessment beyond morphological limitations. • The study informs Middle Pleistocene hominin demography in Iberia. • Ancient protein analysis refines paleoanthropological taxonomic and biological reconstructions.
García‐Martínez et al. (Fri,) studied this question.