he reaction of fluorescamine with 6-aminopenicillanic acid (6-APA) at pH 4 was investigated for its potential use in the biosynthesis of benzylpenicillin. A number of amino acids and penicillin amine derivatives, that reacted with fluorescamine at pH 7-9, were unlikely to do so to a significant extent at pH 4 and hence were unlikely to interfere in results that led to the biosynthesis of benzylpenicillin. Biosynthesis was followed using Penicillium chrysogenum Wis F3-64, growing in a corn steep liquor medium in a shake flask fed with phenylacetic acid daily. Analysis of benzylpenicillin formation was effected enzymically and fluorimetrically. A sample of the fermentation broth was treated with buffer at pH 7.8 and an active penicillin acylase solution for 1 h at 37 °C. The pH was then lowered to 4 by swamping with acetate buffer and the solution was treated with fluorescamine. The resulting fluorescence was compared with that of a standard 6-aminopenicillianic acid solution treated in the same manner. In this case the biosynthesis of benzylpenicillin was found to increase over 6 days. The results were compared with those for a control broth where the penicillin acylase was not added.