Detection of Histoplasma capsulatum urinary antigen (UAg) is important for the initial diagnosis of infection and for monitoring of patient responses to antifungal therapy. This study evaluated an analyte-specific reagent (ASR) enzyme immunoassay (EIA) for the detection of H. capsulatum UAg from Immuno Mycologics, Inc. (IMMY) (Norman, OK) in comparison with routine testing with the MiraVista (MVista) H. capsulatum quantitative EIA (MiraVista Diagnostics, Indianapolis, IN). Using prospectively collected urine specimens (n = 1,003), we observed an overall percent agreement between the two assays of 97.6% (979/1,003 samples). Compared with the MVista EIA, the sensitivity and specificity of the IMMY ASR EIA were 64.5% (40/62 samples) and 99.8% (939/941 samples), respectively, using a cutoff value of 0.5 ng/ml. Based on available clinical histories for 23/24 discordant samples, 5 IMMY assay-negative/MVista assay-positive samples were considered falsely positive. Furthermore, 10/23 discordant samples were positive by the MVista EIA but were below the limit of quantitation (<0.4 ng/ml). The clinical significance of these low positive results in the MVista EIA is unclear. In addition to the prospective study, we tested 11 urine specimens collected from patients with culture-confirmed Histoplasma infections, and 100% (11/11 samples) were positive by the IMMY ASR EIA. In conclusion, the IMMY ASR EIA may offer an alternative approach for the detection of Histoplasma UAg. Additional prospective studies are needed to better characterize the performance of the IMMY ASR EIA in conjunction with clinical and laboratory findings.