Hemocytes from the American lobster (Homarus americanus) were exposed to several receptor-independent and -dependent putative NADPH oxidase stimulators. This stimulation should result in the generation of cytotoxic reactive oxygen species (ROS) that are used by the cells to control infections by destroying ingested microorganisms. Superoxide (O₂⁻) and hypocholorous acid (HOCl) were quantified by the use of chemiluminescent probes. Only phorbol myristate acetate (PMA), of all the stimuli tested, produced a strong ROS response, which was characterized primarily by the generation of a ~20-fold increase in HOCl. Unstimulated cells produced small O₂⁻ and HOCl peaks. Exposure of the cells to arachidonic acid, zymosan, Bacillus subtilis, conA, laminarin and E. coli lipopolysaccharide failed to stimulate net O₂⁻ generation. However, most of these agents as well as PMA caused the time to peak O₂⁻ production to be significantly reduced. The significance of this apparent kinetic shift (KS) in the O₂⁻ is unknown. The results suggest that, although lobster hemocytes in vitro do not respond to a number of known ROS elicitors, PMA will stimulate a large HOCl response presumably involving the typical pathway of protein kinase C activation, translocation of cytosolic NADPH oxidase components to the site of enzyme assembly and cellular activation.