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This work outlines the Italian validation process of the Multidimensional Scale of Attitudes Toward Lesbians and Gay Men, developed by Gato, Fontaine and Carneiro (2012). The tool was administered to a non-probability sample of 960 Italian participants aged between 18 to 88 years and self-identified as heterosexual. A confirmatory factor analysis was conducted and cross-validation strategy was used. Internal validity was tested by measuring the single item reliability, the composite or construct reliability as well as the convergent and discriminant validity. Although the factorial structure of the original model has been replicated, limits arose in the discriminant validity of the factors. An alternative second-order factorial model is then proposed. This model better fits and protects the discriminant validity. Concurrent validity was confirmed by a positive correlation with the Modern Homophobia Scale Revised (Lingiardi, Falanga, & D'Augelli, 2005). Finally, evidence of the instrument's construct validity was obtained by correlational and differential analyses, based on several factors related to homophobia, such as sex, age, educational level, religious and political orientation and contact with homosexual people. Results suggest the applicability of the tool in the Italian context, in order to measure homonegativity, as well as its usefulness in comparing traditional and modern aspects of prejudice.