The presence of maternal complement C3 and Bf in fertilized eggs of zebrafish shown was sensitive to the zebrafish egg cytosol, and that maternal complement involved in the bacteriolytic activity. First, the immune activity was abolished by pre-incubation of anti-C3 antibody with the egg cytosol, a process that would cause the precipitation of the central component of all known complement pathways, C3. Second, the lytic activity was depleted by heating at 45 (a temperature known to inactivate fish complement). To determine which pathway of complement activation might be involved in the bacteriolytic activity of the egg cytosol, the antibodies against C1q (a key component of classical pathway,), C4 (a key component of both classical and lectin pathways) and Bf (a key component of alternative pathway) were utilized to block the special complement pathway, respectively. It was found that precipitation of C1q and C4 causes little loss of the bacteriolytic activity of the egg cytosol, whereas precipitation of Bf results in a significant reduction of the bacteriolytic activity. Furthermore, addition of EGTA to remove Ca²⁺ (necessary for both classical and lectin pathway) from the egg cytosol induces little decrease in the bacteriolytic activity. In contrast, pre-incubation of EDTA to remove both Ca²⁺ and Mg²⁺(necessary for alternative pathway) from the egg cytosol leads to a substantial reduction of the bacteriolytic activity. Taken all the above into consideration, it is found that activation of the alternative pathway is responsible for the bacteriolytic activity of the maternal complement in zebrafish egg cytosol.