The present study aimed to develop a set of sensitive assays to evaluate the presence of different isoforms, the activity degree, and the immunolocalisation of proacrosin-acrosin in sexually mature boars. The goal was to determine the proacrosin-acrosin status of boar spermatozoa throughout epididymal maturation, during ejaculation and after in vitro capacitation. In epididymal samples, proacrosin expression was high in all regions studied. In contrast, α- and β-acrosin expression was low in the caput region, and increased progressively during maturation and in vitro capacitation. In in vitro capacitated samples, the acrosin activity was 2.25 times higher than in the ejaculated samples and immunolocalisation analyses showed redistribution of proacrosin-acrosin at the apical ridge of the head. This study provides relevant data about the expression, localisation and activity of the proacrosin-acrosin system in healthy adult boars that can be used as a base to analyse changes in the proacrosin-acrosin system under pathological conditions.