Purpose The aim of this study is to empirically test the link between employee ambidexterity and two supportive organizational cultures, namely, a perceived culture of empowerment and a knowledge-sharing culture. Furthermore, the paper addresses the mechanisms through which these supportive organizational cultures work to enable employees to engage in ambidextrous behaviour. Specifically, the role of intrinsic motivation is investigated. Design/methodology/approach Data were obtained from 136 managers employed in the five main Belgian service sectors. Findings The findings indicate that a perceived culture of empowerment is positively related to intrinsic motivation, which in turn facilitates employee ambidexterity. Also, a moderating effect of extrinsic motivation on employee ambidextrous behaviour is found. At the same time, a perceived knowledge-sharing culture is having no effect on ambidexterity or intrinsic motivation. Research limitations/implications Insights into the roles of individuals in achieving ambidexterity help to advance the theoretical understanding of how a supportive organizational context may enhance employee ambidexterity. Originality/value Despite the growing body of research on antecedents of ambidexterity, there is still lack of thorough understanding of how a supportive organizational context may enhance employee ambidexterity and the roles of individuals in achieving ambidexterity. This is one of the first studies that investigate these factors in relation to individual level ambidexterity (as opposed to organization level ambidexterity).