The competition between the biodegradable polyhydroxyalkanoates (PHA) and petrochemical plastics could be in terms of cost of production. Also, the impact of PHA on the preservation of the environment has been established in detail. Eorts in the direction of obtaining cheaper raw materials could reduce the cost of production of PHA. Therefore, an experimental design-based medium optimization was carried out for the production of poly-bhydroxybutyrate (PHB) by Azotobacter beijerinckii in shake¯asks. Previous reports have not indicated the application of corn-steep liquor (CSL) as the nitrogen source. Thus attempts were made for the production of PHB using CSL and molasses as the carbon source. It was observed that the combination of molasses and CSL could be used in its production. Analysis of the experimental data has shown the optimum concentration to be 65.4 g/l of molasses and 13.2 g/l of CSL. Also, untreated molasses resulted in a higher yield in comparison with potassiumferrocyanide-treated molasses, indicating the contribution of unidenti®ed components favouring the growth and yield of PHB by Azotobacter beijerinckii.