The kinetics of PaeR7 endonuclease-catalysed cleavage reactions of fluorophor-labeled oligonucleotide substrates have been examined using fluorescence resonance energy transfer (FRET). A series of duplex substrates were synthesized with an internal CTCGAG PaeR7 recognition site and donor (fluorescein) and acceptor (rhodamine) dyes conjugated to the opposing 5' termini. The time-dependent increase in donor fluorescence resulting from restriction cleavage of these substrates was continuously monitored and the initial rate data was fitted to the Michaelis-Menten equation. The steady state kinetic parameters for these substrates were in agreement with the rate constants obtained from a gel electrophoresis-based fixed time point assay using radiolabeled substrates. The FRET method provides a rapid continuous assay as well as high sensitivity and reproducibility. These features should make the technique useful for the study of DNA-cleaving enzymes.