The optimization of P(3HB-co-3HV) synthesis was investigated using response surface methodology (RSM). Primary experiments showed different concentrations of 1-pentanol, agitation rates, and incubation times were the main factors influencing the production of this copolymer. To test the influence of the process parameters, two statistical experimental designs were performed. The first step of optimization used two-level factorial designs to determine the significant variables affecting the growth and production. The RSM model was experimentally validated yielding a maximum of 65 wt % PHA content and 10.2 g/L cell dry weight, which represent 12.1% increase in PHA content in comparison to the non-optimized condition using oleic acid and 1-pentanol as the carbon source and carbon precursor, respectively. The experimental results showed that the RSM was a useful tool as the utilization of 1-pentanol by Cupriavidus sp. USMAA2-4 was reduced by 33%. A second-order polynomial equation was obtained by multiple regression analysis to explain the combined effect of the three parameters with the coefficient of determination, R2, greater than 0.9. The optimum process conditions and interactions were determined by analyzing three-dimensional surfaces. (c) 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012