The conventional means of diagnosis of histoplasmosis presents difficulties because of the delay to the time that the diagnosis is made, indicating the need for the implementation of molecular assays. We evaluated 146 clinical samples from 135 patients suspected of having histoplasmosis using a previously reported nested PCR assay for the Histoplasma capsulatum-specific 100-kDa protein (the Hc100 PCR). In order to determine the specificity of this molecular test, we also used samples from healthy individuals (n = 20), patients suspected of having respiratory disease with negative fungal cultures (n = 29), and patients with other proven infections (n = 60). Additionally, a sizable collection of DNA from cultures of H. capsulatum and other medically relevant pathogens was studied. A panfungal PCR assay that amplified the internal transcribed spacer 2 region was also used to identify all fungal DNAs. All PCR-amplified products were sequenced. Of the 146 clinical samples, 67 (45.9%) were positive by culture and PCR, while 9 samples negative by culture were positive by PCR. All the sequences corresponding to the 76 amplified products presented > or =98% identity with H. capsulatum. The Hc100 PCR exhibited a sensitivity of 100% and specificities of 92.4% and 95.2% when the results were compared to those for the negative controls and samples from other proven clinical entities, respectively; the positive predictive value was 83% and the negative predictive value was 100%; the positive and negative likelihood rates were 25 and 0, respectively. These results suggest that the Hc100 nested PCR assay for the detection of H. capsulatum DNA is a useful test in areas where mycosis caused by this organism is endemic.