Modulation of the respiratory burst activity of head kidney macrophages isolated from rainbow trout (Oncorhynchus mykiss) was observed following treatment with several biologically active substances. Macrophage-activating factor (MAF) induced the highest increment in respiratory burst activity relative to treatment with lipopolysaccharide (LPS), tumor necrosis factor α (TNFα) or β-glucans from Saccharomyces cerevisiae. Increased responses were more evident when these molecules were combined in pairs. Negative regulation of respiratory burst activity was observed when diMePGE 2 was added to the macrophages, with maximal inhibition seen using a concentration of 2.6 μM. Inhibition was also seen using stimulated macrophages, either by co-incubation of stimuli and diMePGE 2 or by adding diMePGE 2 to previously stimulated cells. The inhibitory effect on macrophages was detectable within 3 h of incubation with diMePGE 2 and by 24 h the level of the response was even lower than that from unstimulated (control) macrophages. Of significance was the finding that the inhibitory effect of prostaglandin on macrophage function could be overcome by co-incubation with stimulatory molecules or by pre-treatment with MAF and LPS or MAF and TNFα. Thus, the regulation of macrophage activation in fish is likely to be as complex as in mammals.