In order to raise antibodies for the immunolocalisation and enzyme-linked immunosorbent assay (ELISA)-based detection of the antibiotic tilmicosin, several hapten immunogens have been synthesised and used to produce polyclonal antisera in sheep. Use of the tilmicosin molecule itself as a hapten failed to give a successful immunogen. However, selection of alternative haptens such as desmycosin and lactenocin, which contain reactive aldehyde groups as well as considerable structural similarity to tilmicosin, allowed the preparation of immunogenically active conjugates and, subsequently, anti-tilmicosin polyclonal antibodies. In a microtitration plate competitive ELISA, antisera to desmycosin bound tilmicosin, while cross-reacting with tylosin at 96%. The limit of determination of the ELISA was 0.09 ng ml -1 . Immunoglobulin fractions prepared from the antiserum have also been used for the localisation of tilmicosin in porcine lung by immunocytochemistry and used to determine tilmicosin in avian respiratory tissue. Antiserum to lactenocin showed increased specificity (1.3% cross-reaction with tylosin).