The in vitro effect of levamisole (1, 10 and 100 μg/ml) on cells of the innate immune response of rainbow trout (Oncorhynchus mykiss) was studied, particularly the activity of natural cytotoxic cells and macrophages present in peripheral blood. Levamisole effect on natural cytotoxic cells was evaluated by their capacity to lyse HL-60 tumor cells while activity of macrophages was evaluated measuring production of reactive oxygen species and nitric oxide. Results indicate that natural cytotoxic cells activity was significantly increased by 1 μg/ml levamisole and that higher doses had no effect. On the other hand, reactive oxygen species and nitric oxide production by macrophages was not increased by any of the levamisole doses used and that the higher dose (100 μg/ml) induced a significant decrease of nitric oxide levels, which might be deleterious for the defensive response of these fish.