AbstractThis article focuses on four individuals from France and Italy who were viewed as hermaphrodites and their attempts to become members of the Catholic clergy between c.1650 and 1720. Drawing on largely unexplored material from the archive of the Roman Congregation of the Council, this article argues that whether, and how, bodies were problematised as hermaphroditic depended on the different and changing thresholds of masculinity and femininity they were confronted with. Offering a fresh perspective on practices of constructing sex and sex difference, this article suggests that the decades c.1700 saw marked transformations in the defining and assigning of sex both in theory and social practice. Medical and ecclesiastical decision‐makers shifted their attention from a broader spectrum of behavioural and bodily signs to the anatomy of genitalia. The trend towards heightened vigilance and intransigence towards perceived sexual ambiguity was, however, highly asymmetrical, targeting mainly individuals initially believed to be women.