A variety of supernatants were prepared by stimulating rainbow trout Oncorhynchus mykiss head kidney macrophages with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha), or a leucocyte-derived macrophage-activating factor (I-MAF), individually and in combination. If generated using a 12-h stimulation period, such supernatants were found to elevate significantly the respiratory burst activity of target macrophages; that is, they contained a macrophage-derived MAF (m-MAF), but supernatants generated using a shorter incubation period showed no significant activity. Combinations of these treatments were particularly effective in generating m-MAF-containing supernatants. The elevation of respiratory burst activity by supernatants generated using combined treatments could be partially inhibited by prior treatment of the target macrophages with anti-TNF-alpha receptor 1 (TNFR1) monoclonal antibodies (mAbs). Similarly, treatment of macrophages with combinations of 1-MAF and m-MAF generated supernatants with potent m-MAF activity and this activity was partially inhibited by prior treatment of the target cells with anti-TNFR1 mAb. In addition, the presence of anti-transforming growth factor beta 1 (TGF-beta 1) serum while generating these latter supernatants resulted in significantly increased m-MAF activity. Such data suggest that fish leukocytes secrete a variety of potent macrophage-activating (TNF-alpha) and -deactivating (TGF-beta) factors.