Over the past two decades, the concept of multi-level governance (MLG) has been increasingly discussed by scholars in the fi eld of European integration. While Gary Marks wrote about a four-level arrangement (supranational, national, regional, and local), over time the regional and local levels often became lumped together as 'substate actors' and so easily conceptually interchangeable. This text, however, shows the fallaciousness of this reasoning. In certain circumstances we can fi nd a competitive relationship between cities and regions, positioning themselves against each other for resources and access to national and supranational fora, especially in the context of the new regionalism. The cities have been given substantial support from the European Commission in recent years and we argue that this new constellation may have a remarkable infl uence on relations and possibly also lead to confl icts among local and regional actors in EU multi-level governance. This was possible to be clearly seen in the Czech Republic (CR) between 2012 and 2014, when heated negotiations took place regarding the implementation of the Integrated Territorial Investment (ITI), a fi nancial instrument of EU Cohesion Policy which was implemented on the substate level -i.e., in cities and regions. In the CR this competition took place in a specifi c context, which also infl uenced its outcome.