Cytoplasmic male sterility (CMS) allows efficient biological confinement of transgenes if pollen-mediated gene flow has to be reduced or eliminated. For introduction of CMS maize in agricultural practice, sufficient yields comparable with conventional systems should be achieved. The plus-cultivar-system in maize offers a possibility for biological confinement together with high and stable yields whereas pollinator amount and distribution within the CMS crop is crucial. The aim of this EU-funded study was to identify the best proportion (10, 15, and 20%) and spatial arrangement (inserted rows, mixed seeds) of the pollinator within the CMS maize cultivar under field conditions in the Czech Republic, in Germany and in Spain. In Germany and in the Czech Republic, a pollinator proportion of 10% produced significantly lower yield than the treatments with a pollinator proportion of 15% and 20%. Differences in yield between row and mix arrangements were not detected. No differences between the tested arrangements occurred in Spain. With respect to practical conditions, a pollinator proportion of 15% can be recommended for achieving a satisfactory yield. CMS maize cultivar released no or merely a small amount of pollen and self-pollinated plants developed no or only a small number of kernels indicating that currently recommended isolation distances between genetically modified (GM) and non-GM fields can be substantially shortened if the CMS confinement tool is used