Reactive oxygen species (ROS) production in macrophage-like cells is induced as an antimicrobial defence against invading pathogens. In this study, we have explored how different stimuli and metabolic inhibitors affect the level of respiratory burst in Atlantic salmon (Salmo salar L.) head kidney macrophage-like cells. Cells stimulated in vitro by bacterial lipopolysaccharide (LPS) and ß-glucan showed increased production of ROS compared to unstimulated cells. Both stimulation and costimulation by curdlan (ß-glucan) induced a higher production of ROS compared to stimulation and costimulation by LPS. Metabolic inhibitors co-incubated with the stimulants did not, in most cases, perturb the level of ROS generation in the salmon macrophage-like cells. The NAD content as well as the NAD /NADH ratio increased in curdlan and LPS + curdlan-stimulated cells compared to control cells, which indicated increased metabolic activity in the stimulated cells. Supporting these findings, gene analysis using real-time quantitative PCR showed that arginase-1 and IL-1ß genes were highly expressed in the stimulated cells.