Immunological responses of fishes to glochidia were evaluated using glochidia of the rainbow mussel (Villosa iris) to infest a host species, rock bass (Ambloplites rupestris), and two nonhost species, common carp (Cyprinus carpio) and goldfish (Carassius auratus). Ouchterlony double-diffusion tests showed that host and nonhost species expressed a humoral defense factor specific to glochidial antigens after induced infestation with glochidia. Precipitin bands were observed in tests on infested fishes but not in tests on uninfested fishes. Microagglutination tests showed that host and nonhost species that were uninfested, infested, or reinfested with glochidia all expressed some agglutination response to glochidial antigens. Experimental fishes had specific humoral defense factors that reacted immunologically to glochidia tissue.