To investigate the in vitro generation of nitric oxide (NO) and superoxide anion (O₂^-) by Crassostrea ariakensis hemocytes stimulated with rickettsialike organisms (RLOs), RLOs were purified from infected C. ariakensis. BALB/c mice were infected with the purified RLOs and the infectious vitality of RLOs for the mice was detected using the smears and histological sections stained with Machiavello and transmission electron microscope (TEM) technology. These experiments proved that the purified RLOs retained considerable vitality. The study of the influence of temperature on phagocytosis activity revealed that the percentage of phagocytic cells was lower at 10°C than at 20°C and 30°C. The in vitro generation of the cytotoxic molecules such as NO and O₂^- by C. ariakensis hemocytes was detected when incubated at 25°C with the purified RLOs and zymosan. The generation of O₂^- and NO indicated a possible formation of the highly bactericidal oxidant peroxynitrite by the hemocyte of C. ariakensis. The generation for O₂^- was reduced significantly by superoxide dismutase, but not by inhibitors of NO synthase. Superoxide dismutase and inhibitors of NO synthase decreased the nitrite/nitrate levels generated by C. ariakensis hemocytes. It is suggested that both nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and NO synthase pathways are involved in the self-defense system of C. ariakensis.