In current Bordetella pertussis media ammonium accumulates because of an imbalance in the nitrogen:carbon ratio of the substrates used, which is one of the factors limiting cell density in fed-batch cultures. The aim of this study was to map B. pertussis' catabolic and anabolic capabilities, in order to design a medium that avoids ammonium accumulation, while substrates are metabolised completely. Besides the known dysfunctional glycolysis, B. pertussis also possessed a partially dysfunctional citric-acid cycle. Although ammonium accumulation was avoided by adding various carbon sources to medium with glutamate, nuclear magnetic resonance (NMR) showed excretion of acetate, acetoacetate and b-hydroxy-butyrate, thereby reducing the biomass yield. Acetoacetate and b-hydroxy-butyrate were also formed in Verwey, B2 and modi®ed Stainer ± Scholte medium. Electron microscopy in combination with NMR showed that cells early on in these cultures contained poly-hydroxy-butyrate (PHB) globules, which disappeared later during the culture, coinciding with the appearance of b-hydroxy-butyrate and:or acetoacetate. No globules nor metabolite excretion was detected when lactate in combination with glutamate were used as substrates. Thus, metabolite excretion and ammonium accumulation were avoided, while the yield of 8.8 g C-mol 1 compared favourably with literature values, averaging 6.5 g C-mol 1. Optimisation of this medium for pertussis toxin production will be reported in a separate article. © 1999 Elsevier Science B.V. All rights reserved.