AbstractBackgroundHistoplasmaantigen and anti‐Histoplasmaantibody detection are used to support the diagnosis of histoplasmosis. There is a paucity of published data on antibody assays.ObjectivesOur primary hypothesis was that anti‐Histoplasmaimmunoglobulin G (IgG) antibody detection using enzyme immunoassay (EIA) will be more sensitive as compared to immunodiffusion (ID).AnimalsThirty‐seven cats and 22 dogs with proven or probable histoplasmosis; 157 negative control animals.MethodsResidual stored sera were tested for anti‐Histoplasmaantibodies using EIA and ID. Results of urine antigen EIA were reviewed retrospectively. Diagnostic sensitivity was calculated for all three assays and compared between immunoglobulin G (IgG) EIA and ID. The diagnostic sensitivity of urine antigen EIA and IgG EIA, interpreted in parallel, was reported.ResultsSensitivity of IgG EIA was 30/37 (81.1%; 95% confidence interval [CI], 68.5%‐93.4%) in cats and 17/22 (77.3%; 95% CI, 59.8%‐94.8%) in dogs. Diagnostic sensitivity of ID was 0/37 (0%; 95% CI, 0%‐9.5%) in cats and 3/22 (13.6%; 95% CI, 0%‐28.0%) in dogs. Immunoglobulin G EIA was positive in all animals (2 cats and 2 dogs) with histoplasmosis but without detectable antigen in urine. Diagnostic specificity of IgG EIA was 18/19 (94.7%; 95% CI, 74.0%‐99.9%) in cats and 128/138 (92.8%; 95% CI, 87.1%‐96.5%) in dogs.Conclusion and Clinical ImportanceAntibody detection by EIA can be used to support the diagnosis of histoplasmosis in cats and dogs. Immunodiffusion has an unacceptably low diagnostic sensitivity and is not recommended.