PCR amplification and product analysis for the detection of chromosomal translocations such as bcl-1/JH have traditionally been performed as a two-step process with separate amplification and product detection. PCR product detection has generally entailed gel electrophoresis, hybridization, or sequencing for confirmation of assay specificity. By using a microvolume fluorimeter integrated with a thermal cycler and the PCR compatible double-stranded DNA (dsDNA) binding dye SYBR Green I, we simultaneously amplified and detected bcl-1/JH translocation products by using rapid cycle PCR and fluorescence melting curve analysis. We analyzed DNA from 25 cases of lymphoproliferative disorders comprising 12 previously documented bcl-1/JH-positive mantle cell lymphomas, and 13 reactive lymphadenopathies. The samples were coded and analyzed in a blind manner for the presence of bcl-1/JH translocations by fluorescence melting curve analysis. The results of fluorescence analysis were compared with those of conventional PCR and gel electrophoresis. All of the 12 cases (100%) previously determined to be bcl-1/JH positive by conventional PCR analysis showed a characteristic sharp decrease in fluorescence at about 86šC by melting curve analysis. For easier visualization of melting temperatures (Tm), fluorescence melting peaks were obtained by plotting the negative derivative of fluorescence over temperature (-dF/dT) versus temperature (T). Dilutional assays revealed that fluorescence melting curve analysis was more sensitive than conventional PCR and agarose gel electrophoresis with ultraviolet transillumination by as much as 40-fold. Our results indicate that nucleic acid amplification integrated with fluorescence melting curve analysis is a simple, reliable, sensitive, and rapid method for the detection of bcl-1/JH translocations. The feasibility of specific PCR product detection without electrophoresis or expensive fluorescently labeled probes makes this methodology attractive for studies inmolecular pathology. (Lab Invest 1999, 79:337-345).