FRET (fluorescence resonance energy transfer) probes are random-coiled oligonucleotides containing a reporter at the 5' end and a quencher at the 3' end. Quenching of the FRET probe is achieved by spectral overlap. The use of Black Hole Quencher (BHQ)-labeled FRET probes in real-time polymerase chain reaction (PCR) was evaluated. For S/N measurement, each FRET probe was formulated with a 5' a reporter (FAM) and a 3' quencher (TAMRA, DABCYL, BHQ 1, or BHQ 2). FRET probes were digested with DNase at room temperature for 1 h, and the fluorescence intensities were measured using the LS-50B PCR detection system. The 5'-FAM FRET probe with BHQ 1 at the 3'-end gave the best S/N among the different quenchers evaluated, and TAMRA was the least effective quencher. BHQ-labeled FRET probes could reliably detect target as low as 100 copies, and provide higher sensitivity than TAMRA-labeled probes. They also worked well even under suboptimal PCR conditions.