We have developed an assay procedure to monitor the cleavage of DNA substrates by restriction endonucleases. This procedure uses DNA substrates that are labeled with biotin on one 5' end and with an antigenic group, e.g., fluorescein or digoxigenin, on the other 5' end. After incubation with the restriction enzyme, the reaction is stopped with EDTA and an aliquot is pipetted into the well of an avidin-coated microtiter plate. This immobilizes the unreacted substrate and the biotinylated cleavage product, whereas the other cleavage product labeled with the antigenic group is subsequently washed off. The unreacted substrate is detected by an enzyme-linked immunosorbent assay with an appropriate enzyme-linked antibody. To test our assay we have measured the steady-state rate constants for cleavage of DNA by EcoRI yielding a kcat of 8.6 min-1 and a Km of 150 nM, which are close to values measured with other assays. The advantage of this assay is that it is not only fast and accurate, but also very sensitive. It allows for many samples to be analyzed in parallel and lends itself to automation. Furthermore, this assay can be designed as a competitive assay, when two substrates carrying different antigenic groups are used. The usefulness of such competitive assay is demonstrated by determining the influence of sequence context on the rate of DNA cleavage by EcoRI.