The metabolic fluxes of central carbon metabolism were measured in chemostat-grown cultures of Methylobacterium extorquensAM1 with methanol as the sole organic carbon and energy source and growth limiting substrate. Label tracing experiments were carried out using 70% C-13-methanol in the feed, and the steady-state mass isotopomer distributions of amino acids derived from total cell protein were measured by gas chromatography coupled to mass spectrometry. Fluxes were calculated from the isotopomer distribution data using an isotopomer balance model and evolutionary error minimization algorithm. The combination of labeled methanol with unlabeled CO2, which enters central metabolism in two different reactions, provided the discriminatory power necessary to allow quantification of the unknown fluxes within a reasonably small confidence interval. In wild-type M. extorquensAM1, no measurable flux was detected through pyruvate dehydrogenase or malic enzyme, and there was very little flux through alpha-ketoglutarate dehydrogenase (1.4% of total carbon). In contrast, the alpha-ketoglutarate dehydrogenase flux was 25.5% of total carbon in the regulatory mutant strain phaR, while the pyruvate dehydrogenase and malic enzyme fluxes remained insignificant. The success of this technique with growth on C-1 compounds suggests that it can be applied to help characterize the effects of other regulatory mutations, and serve as a diagnostic tool in the metabolic engineering of methylotrophic bacteria. (C) 2003 Wiley Periodicals, Inc.