ABSTRACT: The capacity of Pasteurella piscicida strains to survive contact with macrophages obtained from rainbow trout Oncorhynchus mykiss, sea bass Dicentrarchus labrax and gilthead sea bream Sparus aurata was evaluated using an in vitro assay. Both virulent and avirulent isolates were killed by all the macrophages tested after 3 and 5 h incubation. The increased production of superoxide anion (O₂^-) by rainbow trout macrophages infected with P. piscicida coinciding with the highest bactericidal activity (5 h incubation) suggests that the O₂^- could be involved in the killing of P. piscicida. Bactericidal activity of cell-free generated O₂^- confirms this hypothesis, although this indicates that some other microbicidal factors must also contribute to the killing of P. piscicida by fish macrophages.