Ascidian sperm were activated and attracted to all of the surface of a whole egg. When the whole egg was separated into follicle cells, chorion and test cells, and the egg itself (naked egg), spermatozoa were activated and exhibited chemotaxis toward the vegetal pole of the naked egg. Such activation and chemotaxis were not observed around isolated follicle cells or chorion with follicle cells and test cells, suggesting that a substance released from the vegetal pole of the egg which activates the sperm and orients them to it in the perivitelline space. The chemotaxis index, which was calculated by a newly proposed method, showed that sperm-attracting activity vanished when the egg deformed, suggesting that the release of attractant through the plasma membrane is terminated at the time of fertilization. The supernatant of the egg suspension (egg seawater) showed strong sperm-activating and sperm-attracting activities, which were dialyzable, heat stable, and resistant to proteinases. Thus, these activities may be initiated by a nonproteinaceous small molecule. Spermatozoa which were previously activated with theophylline without the egg factor showed chemotactic behavior to the tip of the capillary in which egg seawater was enveloped. However, spermatozoa exhibited only activation around the tip of the capillary with theophylline. This indicates that sperm activation and chemotaxis could be controlled under a separative mechanism.