Modelling is an important methodology in systems biology research. In this paper, we presented a kinetic model for the complex ammonium assimilation regulation system of Escherichia coli. Based on a previously published model, the new model included AmtB mediated ammonium transport and AmtB regulation by GlnK. Protein concentrations and several parameter values were determined or refined based on new experimental data. Steady state analysis of the model showed that the expression of AmtB increased the ammonium assimilation rate 4-5-fold at external ammonium concentrations as low as 5 μM. Model analysis also suggested that AmtB and GS levels were coupled to maximize the assimilation flux and to avoid a possible negative ammonia diffusion flux. In addition, model simulation of the short term dynamic response to increased external ammonium concentrations implied that the maximal rate for GlnB/GlnK uridylylation/deuridylylation might be higher for a quick response to environmental changes. © 2009 Elsevier B.V. All rights reserved.