Four commercially available acridinium ester-labeled DNA probes directed against rRNA were evaluated for their ability to identify Blastomyces dermatitidis, Coccidioides immitis, Histoplasma capsulatum, and Cryptococcus neoformans in culture. rRNA was extracted by sonication of 1- to 2-mm2 portions of cultures of fungi in two chaotropic reagents with glass beads. Following a heat inactivation step, the extracts were hybridized in solution with probes specific for each pathogen. The acridinium ester reporter moiety of nonhybridized probe was selectively hydrolyzed, and chemiluminescence of specific DNA:RNA hybrids was quantitated in relative light units with a luminometer. A positive identification required a relative light unit value of > or = 50,000. Sensitivity and specificity of the probes were determined by probing cultures of the respective pathogenic fungi (target) and nontarget fungi. Both mycelial and yeast forms of the dimorphic fungi (B. dermatitidis and H. capsulatum) were tested. For B. dermatitidis, sensitivity and specificity were 87.8 and 100%, respectively (74 target and 219 nontarget fungi tested). For C. immitis, sensitivity and specificity were 99.2 and 100%, respectively (122 target and 164 nontarget fungi tested). For H. capsulatum, sensitivity and specificity were 100 and 100%, respectively (86 target and 154 nontarget fungi tested). For C. neoformans, sensitivity and specificity were 97 and 100%, respectively (100 target and 230 nontarget fungi tested). For B. dermatitidis, C. immitis, and C. neoformans, repeat testing increased the respective sensitivities to 97.3, 100, and 100%. The high sensitivities and specificities of the probes, the relatively short time (less than 1 h) required to perform the assay, and the availability of standardized reagent kits make the acridinium ester-labeled DNA probes well suited to laboratories in need of a rapid method to identify these fungal pathogens. Further, use of the probes to identify pathogenic fungi as soon as colonies appear on primary recovery media significantly shortens the time to reporting.