The hemocytes from the giant freshwater prawn Macrobrachium rosenbergii were examined for their ability to generate superoxide anion (O 2
À
) in vitro upon exposure to various components derived from microbial cell wall components. Among the test molecules, laminarin (a polymer of b-1, 3 glucans), mannan and LPS from five different bacterial species produced a differential response in terms of stimulated O 2 À production in prawn hemocytes, suggesting the ability of the hemocytes to differentiate non-self. This response was almost completely inhibited by superoxide dismutase (SOD) suggesting SOD-inhibitable O 2 À generation by prawn hemocytes. Phorbol 12-myristate 13-acetate (PMA) and Ca ionophore led to enhanced O 2 À generation by the hemocytes and this suggests the possible role of protein kinase C and Ca 21 ions in such generation. Cytochemical analysis using nitroblue tetrazolium (NBT)-staining revealed the importance of granular hemocytes in O 2 À generation in these prawns. Inhibition of O 2 À generation by inhibitors of NADPH-oxidase and phenoloxidase pathways clearly reveal the involvement of two different pathways in non-self stimulated O 2 À generation by the prawn hemocytes. These findings demonstrate the importance of O 2 À generation and role of possible pathways in hemocyte mediated cellular immune response of a crustacean.