Streptomyces niveus in a chemically defined medium requires the inclusion of Krebs cycle acids for good growth. These acids are used in preference to glucose, and this results in diauxic growth. Studies with washed cells, cell-free extracts, and mutants of S. niveus were performed to determine the mode of synthesis of the individual ring systems of novobiocin and their linkage. A pathway for novobiocin synthesis is proposed on the basis of these experiments.