Some marine coenocytic green algae could form protoplasts from the extruded protoplasm in seawater. The dissociated cell components of the coenocytic protoplasm could be reunited into live cells and, hence, the formation of new species by mixing protoplasms from different coenocytic cells has been predicted. Our results showed that an incompatibility barrier was present during protoplast formation in coenocytic algae to exclude foreign inorganic particles or alien cell components. No inorganic particles or alien cell components were incorporated into protoplast formed spontaneously in seawater. Even when the inorganic particles or alien cell and/or cell component were incorporated into protoplast in some experimental condition, they were expelled from the protoplast or degenerated within several days. A species-specific cytotoxicity was observed during protoplast hybridization between the protoplasms of Bryopsis spp. and Microdictyon umbilicatum. The cell sap of M. umbilicatum could destroy the cell components of Bryopsis spp., but had no effect on Chaetomorpha moniligera. Species C. moniligera and Bryopsis did not affect protoplast generation of either species. The wound-induced protoplast formation in vitro might have evolved in some coenocytic algae as a dispersal method, and the incompatibility barrier to alien particles or cell and/or cell component could serve as a protective mechanism for successful propagation.