Immunological assays were developed to test for the effects of immunisation with Aeromonas salmonicida O antigen bacterin in rainbow trout (Oncorhynchus mykiss) in vitro and in vivo. Trout spleen sections were immunised in vitro by incubation in media containing 100, 10, and 1 μg ml⁻¹ of the A. salmonicida O antigen. After 10 days at 15°C, the individual sections were minced and cell suspensions assayed for the effects of the bacterin on non-specific and specific immune responses. Assays of the cell suspensions showed that the neutrophil oxidative activity, phagocytic capabilities, numbers of plaque-forming cells (PFC) and circulating antibody titres all increased in proportion to the bacterin dosage. Trout immunised in vivo with 100 μg by injection and by 100 μg ml⁻¹ bath with the O antigen, showed similar responses to spleen sections immunised in vitro. The appearance of non-specific PFC against unlabelled sheep red blood cells was highest in spleens immunised in vitro and in the fish immunised by injection; non-specific PFC rarely occurred in O antigen bathed fish and in control fish. The development of these assays may enable biologists to follow the effects on the non-specific defence mechanisms and the specific immune response in cultivated fish populations after, immunisation with A. salmonicida bacterins and thus aid in the development of protective vaccination against furunculosis.