Metabolomics emerges to become an important profiling technique in bio(techno)logical systems. In addition to intracellular metabolite concentrations at (quasi) stationary conditions, stimulus-response experiments provide information on the dynamic behavior of metabolic pathways. These data are relevant for bioprocess analysis on the level of metabolism and for application of metabolic engineering principles aiming at a metabolic redesign of producer cells. However, even for the well-studied bacteria Escherichia coli only limited growth-rate dependent intracellular metabolite information is currently available, thereby impeding comprehensive metabolome analysis. Here, we present intracellular metabolite concentration data of representative glycolytic intermediates in E. coli cultivated in glucose-limited chemostats, (i) at systematic variation of growth-rate (D = 0.1, 0.2, 0.3, and 0.4 h−1) and (ii) at both steady-state and after a glucose pulse applying a recently introduced integrated sampling procedure and LC-MS analytical method. Whereas intracellular steady-state concentrations of upper part glycolytic intermediates FBP and DHAP+GAP increased 2.3-fold, respectively 2.8-fold, when specific growth-rate is raised from μ = 0.1 h−1 to μ = 0.4 h−1, the opposite trend was observed for 2PG+3PG and PEP pools with a decrease by a factor of 2.1, respectively 1.9. In glucose pulse experiments FBP and DHAP+GAP showed a 3.3 (1.8)-fold, respectively 2.8 (2.0)-fold, increase relative to the steady-state level at μ = 0.1 (0.4) h−1. Also, the dynamics changed with growth-rate for these two metabolite pools. In contrast, 2PG+3PG and PEP were characterized by decreased concentrations in response to a glucose pulse and the relative changes related to steady-state values were significantly smaller compared with FBP and DHAP+GAP. The observed growth-rate dependency of our data clearly indicates the necessity for metabolome studies covering a broader range of physiological growth conditions.