As part of ongoing studies in the biosynthesis of the lantibiotic nisin, we investigated the proteolytic cleavage of a synthetic Fmoc-labeled decapeptide mimicking a key amino acid sequence of the precursor prenisin in extracts of a Lactococcus lactis subsp. lactis strain. Reverse-phase high-performance liquid chromatography with photodiode army detection was used to trace and purify potential enzymatic conversion products. Of the three newly appearing chromatographic peaks, one was identified by means of electrospray mass spectrometry, amino acid analysis. and amino acid sequencing as an Fmoc-labeled hexapeptide derived from cleavage at the Arg(-1)-Ile(+1) bond. This assay will be useful to monitor the purification of the endoproteinase that reportedly cleaves prenisin at the same Arg(-1)-Ile(+1) site present in the model substrate.