Actinophryid and centrohelid heliozoans often share the same habitat; however, field studies have shown that they preferentially exist separately than together, suggesting the existence of a mechanism that does not allow them to co-exist. Therefore, to clarify the interactions between these heliozoans, co-culturing experiments, involving two organisms, an actinophryid and a centrohelid, were performed. For the experiments in the brackish water environment, Actinophrys sol (actinophryid) and Raphidocystis contractilis (centrohelid) were used, and for those in the freshwater environment, a combination of Actinosphaerium eichhornii (actinophryid) and one of three centrohelid species (Choanocystis pantopoda, Raphidocystis ambigua, or Raphidocystis marginata) were used. In all the experiments involving the co-culture environments, the centrohelids grew intensively within the first 3-5 days, after which their cell numbers remained constant. Conversely, the number of actinophryid heliozoans decreased significantly and their disappearance was recorded after 2 days and after 5-11 days for the experiments in the brackish water and freshwater environments, respectively. Further, in some experiments, several centrohelid cells fused together to form a giant multinucleated cell, which jointly, captured and consumed larger organisms, such as Actinophrys sol. Furthermore, Actinosphaerium eichhornii (actinophryid) exhibited an avoidance response against Choanocystis pantopoda (centrohelid) attack. Thus, our experimental results showed that in mixed co-culture environments, centrohelids inhibit the growth of actinophryids. This possibly explains the exclusive mode of existence of these two groups of heliozoans in the natural environment.