We used fungus-specific PCR primers and species-specific DNA probes to detect up to three Candida species in a single reaction tube by exploiting the 5' to 3' exonuclease activity of Taq DNA polymerase. Probes to the internal transcribed spacer region of the rRNA gene were labeled at the 5' end with one of three fluorescent reporter dyes, 6-carboxy-fluorescein (FAM), tetrachloro-6-carboxy-fluorescein (TET), or hexachloro-6-carboxy-fluorescein (HEX), and at the 3' end with a quencher dye, 6-carboxy-tetramethyl-rhodamine. During PCR amplification, each reporter dye emits a characteristic wavelength as it is cleaved from its specific target DNA and from the quencher dye. Therefore, signals from up to three probes can be detected simultaneously during the PCR assay. Six probes were designed for use in this study: CA-FAM, CT-TET, and CP-HEX were added to one tube to simultaneously detect the typically fluconazole-sensitive species C. albicans, C. tropicalis, and C. parapsilosis, respectively. CG-FAM and CK-TET were added to a second tube to simultaneously detect the typically more innately fluconazole-resistant species C. glabrata and C. krusei, respectively. All-CAN-TET, a Candida genus probe, was added to a third tube to detect DNAs from all Candida species tested. DNAs recovered from 61 blood culture bottles, including 23 positive for C. albicans, 18 positive for C. glabrata, 6 positive for C. tropicalis, 6 positive for C. krusei, 5 positive for C. parapsilosis, and 3 positive for mixed fungemias, were tested. Control samples included those from blood culture bottles with no growth (n = 10) or from patients with confirmed bacteremia (n = 10). Probes detected and correctly identified the organisms in 58 of 61 specimens (95.1%) and gave no false-positive results. This method is simple and rapid and does not require post-PCR hybridization and incubation steps. It is sensitive and specific for the detection and identification of Candida species from blood culture bottles, including those containing mixtures of Candida species, and should facilitate an earlier specific diagnosis, leading to more appropriately targeted antifungal drug therapy.