A fluorescence resonance energy transfer (FRET) study of biomolecules typically employs two fluorophores. The increasing number of branches and complexity of biomolecules call for simultaneously monitoring structures and dynamics of several branches in a single system. Furthermore, despite recent studies that show DNAzymes can be a stable and cost-effective alternative to protein and ribozymes for pharmaceutical and biotechnological applications, no FRET study of DNAzymes has been reported. Here, we describe the FRET study of a trifluorophore-labeled "8-17" DNAzyme, in which each of the three branches is labeled with a different fluorophore. From the study, we found that the (ratio)A method that has been commonly used in dual-fluorophore-labeled systems is also applicable to trifluorophore-labeled systems. However, while both FRET efficiency and fluorophore-to-fluorophore distance can be used to measure FRET in dual-fluorophore-labeled systems, only the average distance should be used in trifluorophore-labeled systems. The ability to monitor all three branches in a single system allowed us to reveal new metal-ion-dependent conformational changes in the DNAzyme. The trifluorophore-labeled "8-17' DNAzyme has been found to adopt a two-step folding process in the presence of Zn2+. Each step is induced by one Zn2+ binding, with apparent dissociation constants of 19 muM and 260 AM for binding the first and second Zn2+ , respectively. The trifluorophore FRET results are verified by a dual-labeled control experiment. The results demonstrated that the trifluorophore-labeled system is simple and yet powerful in studying complicated biomolecular structure and dynamics and is capable of revealing new sophisticated structural changes that may have functional implications.