A streptogramin type B antibiotic, virginiamycin S (VS), is produced by Streptomyces virginiae, together with a streptogramin type A antibiotic, virginiamycin M1 (VM), as its synergistic counterpart. VS is a cyclic hexadepsipeptide containing a nonproteinogenic amino acid, Lphenylglycine (L-pheGly), in its core structure. We have identified, in the left-hand extremity of the virginiamycin supercluster, two genes that direct VS biosynthesis with L-pheGly incorporation. Transcriptional analysis revealed that visF, encoding a nonribosomal peptide synthetase, and visG, encoding a protein with homology to a hydroxyphenylacetyl-CoA dioxygenase, are under the transcriptional regulation of virginiae butanolide (VB), a small diffusing signalling molecule that governs virginiamycin production. Gene deletion of visG resulted in complete loss of VS production without any changes in VM production, suggesting that visG is required for VS biosynthesis. The abolished VS production in the visG disruptant was fully recovered either by the external addition of pheGly or by gene complementation, which indicates that VisG is involved in VS biosynthesis as the provider of an L-pheGly molecule. A feeding experiment with L-pheGly analogues suggested that VisF, which is responsible for the last condensation step, has high substrate specificity toward L-pheGly.