In established methods for analyzing ribozyme kinetics, radiolabeled RNA substrates are primarily used. Each data point requires the cumbersome sampling, gel electrophoretic separation, and quantitation of reaction products, apart from the continuous loss of substrate by radioactive decay. We have used stable, double fluorescent end-labeled RNA substrates. Fluorescence of one fluorophore is quenched by intramolecular energy transfer (FRET). Upon substrate cleavage, both dyes become separated in two RNA products and fluorescence is restored. This can be followed in real time and ribozyme reactions can be analyzed under multiple (substrate excess) and under single (ribozyme excess) turnover conditions. A detailed comparison of unlabeled, single, and double fluorescent-labeled RNAs revealed moderate kinetic differences. Results with two systems, hammerhead ribozymes in I/II (small ribozyme, large substrate) and in I/III format (large ribozyme, small substrate), are reported.