Egg-derived sperm-activating factors and attractants activate sperm motility and attract the sperm, respectively. These phenomena constitute the first communication signaling between males and females in the process of fertilization in many animals and plants, and in many cases, these are species-specific events. Thus, sperm motility activation and chemotaxis may act as a safety process for the authentication between conspecific egg and sperm, and help to prevent crossbreeding. Here, we examine species-specificity of sperm motility activation and chemotaxis in the ascidians belonging to the order Phlebobranchiata: Ciona intestinalis, Ciona savignyi, Phallusia mammillata, Phallusia nigra, and Ascidia sydneiensis. Cross-reactivity in both motility activation and chemotaxis of sperm was not observed between C. savignyi and P. mammillata, or between A. sydneiensis and Phallusia spp. However, there is a "one way" (no reciprocity) cross-reaction between P. mammillata and P. nigra in sperm activation, and between C. savignyi and A. sydneiensis in sperm chemotaxis. Furthermore, the level of activity is different, even when cross-reaction is observed. Thus, sperm motility activation and chemotaxis are neither "species-" nor "genus-" specific phenomena among the ascidian species. Moreover, the interaction between the sperm-activating and sperm-attracting factors (SAAFs) in the ascidian species and the SAAF receptors on the sperm cells are not all-or-none responses.