The production of polyhydroxybutyrate (PHB) and extracellular polymeric substances (EPS) by Ralstonia eutropha ATCC 17699 at various glucose and (NH4)2SO4 concentrations in batch cultures were investigated. The biosynthesis of EPS by R. eutropha closely coupled with cell growth, while PHB was synthesized only under nitrogen-deficient and cell-growth-limited conditions. Experimental results show that the specific PHB production rate had an exponential correlation with both specific cell growth rate and EPS production rate. Furthermore, PHB was observed as the main storage of carbon and energy source by R. eutropha under nitrogen-limited conditions. In addition, experiments were conducted based on central composite design to optimize the batch culture for a high PHB yield. The PHB yield on glucose reached a maximum value of 0.34 g/g at glucose concentrations of 38.2 g/l and (NH4)2SO4 of 3.2 g/l.