Polyhydroxyalkanoates are biodegradable polymer materials that accumulate in numerous bacteria. The polyhydroxybutyrate is the most common type of polyhydroxyalkanoates, which potentially serves as precursor for bioplastic production. The most extensively studied polyhydroxybutyrate producing bacteria is Cupriavidus necator due to its capability to accumulate large amounts of this biopolymer in simple culture medium. Accumulation of polyhydroxyalkanoates granules in the cytoplasm of C. necator significantly depended on pH, aeration, carbon sources, nitrogen sources, and minerals in the culture medium. In the present study, the effect of both nutritional and physical variables on polyhydroxybutyrate production was investigated in order to optimize these conditions. At first, on the basis of one-factor-at-a-time experiments, fructose and ammonium chloride were found to be the most suitable sources of carbon and nitrogen for biopolymer production. Then the most significant factors affecting granules accumulation were recognized as fructose, agitation speed, KH 2 PO 4 , and initial pH using the Plackett-Burman and central composite design. ANOVA analysis showed significant interaction between fructose and agitation speed. After optimization of the medium, compositions for polyhydroxybutyrate production were determined as follows: fructose 35 g/L, KH 2 PO 4 1.75 g/L, MgSO 4 Á7H 2 O 1.2 g/L, citric acid 1.7 g/L, trace element 10 mL/L, initial pH = 7, and agitation speed 175 rpm. Under this optimal culture conditions, the maximum yield of PHB was 7.48 g/L. The present strategies included in this study could be used for PHB production by this bacterium. These results are the highest values of PHB ever obtained from batch culture of C. necator reported so far.