Bacilysin is a dipeptide antibiotic composed of L-alanine and L-anticapsin. We recently reported partial purification and characterization of the bacilysin synthetase. In the present study, L-anticapsin in the in vitro reaction mixture was replaced by furfurylamine, norephedrine, glycine, and benzylamine, respectively. The enzymatic biosynthesis of novel structures was checked by TLC analyses of reaction mixture extracts and any positive TLC result was verified by GC-MS analysis. The enzyme was shown to link furfurylamine to L-alanine, hence synthesizing 2-amino-N-(2-furylmethyl)propanamide, but did not act on the other substrates tested to replace anticapsin. (C) 2003 Elsevier Inc. All rights reserved.