Immunoenhancing activity of synthetic muramyl dipeptide (MDP) was investigated in relation to the activation of rainbow trout (Oncorhynchus mykiss) phagocytes and to nonspecific protection against challenge with the fish pathogen Aeromonas salmonicida. Head kidney phagocytes collected from MDP-injected fish showed significant chemotactic activity against zymosan-activated normal rainbow trout serum, and phagocytic activities against both A. salmonicida and plastic beads. The MDP-activated phagocytes also showed enhanced superoxide generation. When normal phagocytes were exposed to supernatants of phagocyte or peripheral blood lymphocyte cultures of MDP-injected fish, the cells showed significant chemotactic activity, indicating that the MDP-activated cells produced phagocyte-activating factor. Injection of MDP to fish provided protection against challenge with virulent A. salmonicida.