We have developed MetaFluxNet which is a stand-alone program package for the management of metabolic reaction information and quantitative metabolic flux analysis. It allows users to interpret and examine metabolic behavior in response to genetic and/or environmental modifications. As a result, quantitative in silico simulations of metabolic pathways can be carried out to understand the metabolic status and to design the metabolic engineering strategies. The main features of the program include a well-developed model construction environment, user-friendly interface for metabolic flux analysis (MFA), comparative MFA of strains having different genotypes under various environmental conditions, and automated pathway layout creation. The usefulness and functionality of the program are demonstrated by applying to metabolic pathways in E. coli. First, a large-scale in silico E. coli model is constructed using MetaFluxNet, and then the effects of carbon sources on intracellular flux distributions and succinic acid production were investigated on the basis of the uptake and secretion rates of the relevant metabolites. The results indicated that among three carbon sources available, the most reduced substrate is sorbitol which yields efficient succinic acid production. The software can be downloaded from http://mbel.kaist.ac.kr/.