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Lateralization is a key aspect of brain architecture and handedness is its primary manifestation. The Edinburgh Handedness Inventory (EHI) and the laterality quotient (LQ) assess the direction and consistency of handedness and require translation and cross-cultural adaptation to guarantee construct validity. We developed a standardized Italian EHI version. The developed Italian version was tested on 202 Italian subjects, classified into three hand types based on their LQs: right, mixed, and left. The frequency of left-handedness in Italians and other populations was compared to previous data. LQs from the twenty- and the ten-item original inventories were also compared. We conducted a factorial analysis. Mcdonald's Omega tested internal consistency. The prevalence of left-handedness was 6.4%, consistent with prior findings in Italian samples and other EHI translations. Age was the only socio-demographic variable that significantly affected the LQ. The internal consistency of the Italian EHI was excellent. Handedness is a feature of several cognitive functions and some neuropsychological diseases; it is influenced by socio-demographic and cultural factors and the instrument used to assess it. To provide a consistent and comparable evaluation of the construct, we recommend using this validated Italian translation of the EHI.