Erysipelothrix rhusiopathiae, a Gram positive, rod-shaped bacteria produces a disease (erysipelas) which in bottlenose dolphins (Tursiops truncatus) may be rapidly fatal. Availability of a simple, reliable, and sensitive assay for measuring levels of antibody against, E. rhusiopathiae is important for determining past exposure to the agent, immune status, and effectiveness of developmental vaccines against the agent. Use of an enzyme linked immunosorbent assay (ELISA) with a "double indirect" indicator antibody system (unlabeled rabbit anti-dolphin immunoglobulin followed by alkaline phosphatase (AP)-labeled anti-rabbit IgG) for determination of the level of antibodies against E. rhusiopathiae in dolphin serum samples has previously been reported by other investigators. In the present study, a biotin/avidin amplified ELISA is reported in which the indicator system consisted of (1) anti-dolphin immunoglobulin antibodies labeled with biotin and (2) avidin labeled AP. Para-nitrophenyl phosphate (pNP) was used as the chromogenic indicator substrate. Indicator antibody preparations employed included biotin labeled rabbit anti-dolphin serum and biotin conjugated rabbit anti-dolphin IgG (where the IgG immunogen had been purified by protein G chromatography). The capture antigen preparation (used to coat plates used in the assay) consisted of extracted surface antigens from a culture of E. rhusiopathiae isolated at the Naval Oceanographic Systems Center (NOSC). Serum samples (provided by NOSC) from 97 dolphins (many of which had been previously immunized with various E. rhusiopathiae vaccine preparations) were assayed and compared with a negative reference serum for level of E. rhusiopathiae antibodies. The dolphin serum samples were assayed as "blind" samples. Assays using labeled anti-purified dolphin IgG yielded endpoints suggesting sensitivities at least twice that of assays using anti-dolphin serum. l he biotin/avidin indicator system provided reliably duplicable labeled indicator antibodies, as well as providing increased sensitivity compared to that obtained using AP labeled antibodies.