For the newly isolated chemoheterotrophic bacterium Citrobacter amalonaticus Y19, anaerobic glucose metabolism and hydrogen (H(2)) production pathway were studied using batch cultivation and an in silico metabolic-flux analysis. Batch cultivation was conducted under varying initial glucose concentration between 1.5 and 9.5 g/L with quantitative measurement of major metabolites to obtain accurate carbon material balance. The metabolic flux of Y19 was analyzed using a metabodic-pathway model which was constructed from 81 biochemical reactions. The linear optimization program MetaFluxNet was employed for the analysis. When the specific growth rate of cells was chosen as an objective function, the model described the batch culture characteristics of Ci. amalonaticus Y19 reasonably well. When the specific H(2) production rate was selected as an objective function, on the other hand, the achievable maximal H(2) production yield (8.7 mol H(2)/mol glucose) and the metabolic pathway enabling the high H(2) yield were identified. The pathway involved non-native NAD(P)-linked hydrogenase and H(2) production from NAD(P)H which were supplied at a high rate from glucose degradation through the pentose phosphate pathway (c) 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.