Assessment of the immune system's capability to respond to antigens with the generation of specific antibodies, whilst under the influence of a test article, is required in toxicity tests according to the European guideline for repeated dose toxicity testing of medicinal products. The purpose of this study in rats was to validate methodology for the determination of Keyhole Limpet Haemocyanin (KLH)-specific antibodies under the influence of an immunologically active compound. The immunosuppressant FK506, commercially available as Prograf, was administered orally (gavage) to five rats per sex per group at dosages of 0.5mg/kg per day or 3mg/kg per day, for a period of 4 weeks. On days 14 and 22, KLH was administered subcutaneously, with an adjuvant (AluGel), to the two treated groups and a control (i.e. without FK506 treatment) approximately 1h following administration of FK506. Terminal investigations included haematology parameters, titration of KLH-specific antibodies in serum (ELISA), macroscopic pathology, spleen and thymus weights, immunophenotyping of splenocytes (FACS analysis) and histopathology of the lymphatic tissues. At 3mg/kg per day a minimal reduction of subcutaneous KLH-induced granuloma formation and a moderate to marked reduction of germinal centre development (axillary lymph node and spleen) were observed. Reduced CD4+ (T-cell) counts were found in the spleen of males, consistent with a suppressed production of KLH-specific antibodies (IgG in both sexes, IgM in males only) and a higher incidence of atrophy in the periarteriolar lymphoid sheaths of males. Slight-to-moderate lymphopenia was present in both sexes at 3mg/kg per day. These findings are consistent with the known pharmacological activity of FK506. In conclusion, determination of antibody titres following immunisation of rats with KLH, with concurrent exposure to a drug, appears to be a valid method in the context of the immunotoxicity evaluation required by European regulation.