Proteome analysis using two-dimensional electrophoresis (2-DE) was applied to characterize the physiological states of Brevibacterium flavum during threonine-limited lysine fermentation. Cell samples taken from batch culture under different physiological states were analyzed by 2-DE, and protein spots were identified by peptide mass fingerprinting. On differential analysis of 2-DE maps, the differences in physiological states of microorganisms were recognized at the protein level. The proteomic information obtained by 2-DE of culture samples provided a global and dynamic view of changes in the physiological state of B. flavum in response to environmental conditions.