Pertussis toxin (Ptx) expression and secretion in Bordetella pertussis are regulated by a two-component signal transduction system encoded by the bvg regulatory locus. However, it is not known whether the metabolic pathways and growth state of the bacterium influence synthesis and secretion of Ptx and other virulence factors. We have observed a reduction in the concentration of Ptx per optical density unit midway in fermentation. Studies were conducted to identify possible factors causing this reduction and to develop culture conditions that optimize Ptx expression. Medium reconstitution experiments demonstrated that spent medium and a fraction of this medium containing components with a molecular weight of <3,000 inhibited the production of Ptx. A complete flux analysis of the intermediate metabolism of B. pertussis revealed that the sulfur-containing amino acids methionine and cysteine and the organic acid pyruvate accumulated in the media. In fermentation, a large amount of internal sulfate (SO₄²) was observed in early stage growth, followed by a rapid decrease as the cells entered into logarithmic growth. This loss was later followed by the accumulation of large quantities of SO₄² into the media in late-stage fermentation. Release of SO₄² into the media by the cells signaled the decoupling of cell growth and Ptx production. Under conditions that limited cysteine, a fivefold increase in Ptx production was observed. Addition of barium chloride (BaCl₂) to the culture further increased Ptx yield. Our results suggest that B. pertussis is capable of autoregulating the activity of the bvg regulon through its metabolism of cysteine. Reduction of the amount of cysteine in the media results in prolonged vir expression due to the absence of the negative inhibitor SO₄². Therefore, the combined presence and metabolism of cysteine may be an important mechanism in the pathogenesis of B. pertussis.