Publisher Summary This chapter discusses the mechanism of penicillin biosynthesis. Penicillin is produced by strains of Penicillium notatum or Penicillium chrysogenum when inoculated into a suitable nutrient medium. The biosynthesis of benzyl penicillin occurs via the condensation of its precursors, phenyl acetic acid, L-cysteine, and L-valine. The rate-limiting reaction is the synthesis of the side-chain precursor, and thus phenyl acetate is usually added to the fermentation medium. Acylation of the β-lactam-thiazolidine ring appears to be one of the final steps in biosynthesis. A large number of compounds are capable of being incorporated into the cysteine moiety of penicillin. Added L-cystine (after conversion to L-cysteine) is used as an intact molecule for penicillin synthesis. The incorporation is stereo specific for the L-form, which is the configuration present in penicillin. The remaining portion of the molecule is synthesized from t-valine despite the fact that the valine moiety of penicillin is of D-configuration. Results of several studies have indicated that the intact carbon skeleton of valine is used, but the amino group is lost during biosynthesis.