We evaluated the side effects induced by injection of Freund's adjuvant (FA) and alternative adjuvants combined with different antigens. Rabbits and mice were injected subcutaneously, intramuscularly (rabbits) and intraperitoneally (mice) with different adjuvants (FA, Specol, RIBI, TiterMax, Montanide ISA50) in combination with several types of antigens (synthetic peptides, autoantigen, glycolipid, protein, mycoplasma or viruses). The effects of treatment on the animals' well-being were assessed by clinical and behavioural changes (POT and LABORAS assays) and gross and histopathological changes. In rabbits, treatment did not appear to induce acute or prolonged pain and distress. Mice showed behavioural changes immediately after (predominantly secondary) immunization. Injection of several adjuvant/antigen mixtures resulted in severe pathological changes, depending on adjuvant, type of antigen, animal species used and route of injection. Both rabbits and mice showed pathological changes ranging from marked to severe after injection of FA, and ranging from minimal to marked after Specol and Montanide injections. Pathological changes after RIBI injections were severe in rabbits, though slight in mice. After TiterMax injections, pathological changes were moderate in rabbits, though severe in mice. In conclusion, injection of FA according to present guidelines resulted mostly in severe pathological changes, whereas only very few clinical and behavioural signs indicated prolonged severe pain. Our findings indicate that Montanide ISA50 and Specol induce acceptable antibody titres, and cause fewer pathological changes than FA. Thus they are effective alternatives to FA.