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.