Bacillus subtilis NX-2 produces gamma-polyglutamic acid (gamma-PGA) when using glucose and L-glutamate as carbon sources. The conversion of carbon sources into gamma-PGA was analyzed with the C-13-NMR method after enriching the media with C-13-labeled glucose. The results showed that the percentage of gamma-PGA monomers derived from glucose was relatively low, approximately 6% and 9%, respectively, with an initial glucose concentration of 30 and 40 g L-1. It was concluded that glucose was utilized mainly as the growth-limiting substrate for cell growth and supplied the required energy during gamma-PGA biosynthesis, while L-glutamate was preferred as the main substrate for gamma- PGA formation. To achieve an efficient conversion of L-glutamate and enhance the gamma-PGA production, a fed-batch culture was proposed by feeding of glucose. By this method, supplied L-glutamate (40 g L-1) was completely depleted, and gamma-PGA yield was attained 42 g L-1.