A biotin-avidin amplified enzyme linked immunosorbent assay (ELISA) for the detection of antibodies against Erysipelothrix rhusiopathiae was developed utilizing a "double indirect" indicator antibody system (biotin-labeled rabbit anti-T. truncatus immunoglobulin followed by avidin-labeled alkaline phosphatase). Marine mammal sera were obtained from Dr. William Van Bonn at Naval Research and Development. Sera from Atlantic bottlenose dolphins (Tursiops truncates) comprised the majority of the samples, but some beluga whale (Delphinapterus leucus) and Pacific bottlenose dolphin (Tursiops gill) samples were also assayed. Samples from animals at Marine Animals Productions in Gulfport, MS (T. truncates), and John G. Shedd Aquarium in Chicago, IL (beluga whale and Pacific white-sided dolphin (Lagenorhyncus obliquidens) were also assayed by the ELISA protocol. A micro-agglutination protocol was also employed to provide a basis for comparison of the results of the ELISA to titers determined by classical serological assay. In direct micro-agglutination assays, suspensions of E. rhusiopathiae were diluted to 40% transmittance at 600 nm in normal phosphate buffered saline (PBS). Doubling dilutions were made of the serum samples in Terasaki micro-well plates to a volume of 5 µl, and an equal amount of the cell suspension was added. After a one hour incubation at 37°C, agglutination was scored (0 - 4+) using a phase contrast, inverted light microscope. Controls included wells with PBS only, PBS and cells only, PBS and serum sample only and a positive control using antisera against E. rhusiopathiae rose in rabbits. For the procedure, titer can be defined as the highest dilution displaying any agglutination, at least 2+ agglutination, or complete (4+) agglutination. Correlation between the ELISA and agglutination results is highest when using an ELISA titer definition of the mean absorbance of the sample minus two standard deviations compared to negative serum, and an agglutination titer defined as the highest dilution exhibiting 2+ agglutination. Using this definition, serum samples with ELISA titers between 1/100 and 1/1000 showed agglutination titers, with the median titer being between 1/64 and 1/128. Less correlation was found for samples with ELISA titers of < 1/100 (the lowest dilution employed in the ELISA's reported here). ELISA's performed using lower and more closely spaced dilutions should address this potential limitation to the use of ELISA's.