2,3,5,6-Tetramethylpyrazine (TTMP) was produced using a newly isolated Bacillus mutant. Culture medium optimization studies showed that soytone, an enzyme-hydrolysate of soybean meal, with the supplementation of vitamins, can fully replace yeast extract plus peptone in supporting TTMP production from glucose. In a 5-l fermenter, using the optimized medium which contained 20% glucose, 5% soytone, 3% (NH(4))(2)HPO(4), and vitamin supplements, fermentations were carried out with stirring at 700 rpm, air flow at 1.0 vvm, controlled pH at 7.0, and temperature at 37 degrees C. TTMP reached 4.33 g l(-1) after 64.6 h cultivation. A product recovery method was described, which involved evaporation, crystallization, and lyophilization. The product purity was 99.88%, determined by GC with the normalization method. The main impurities were 2,3,5-trimethylpyrazine (0.09%) and 2-ethyl-3,5,6-trimethylpyrazine (0.02%), which were identified by GC/MS. (13)C NMR determination also gave a consistent result. Natural and high purity of the product and the utilization of cheap green renewable materials make this process promising to compete with TTMP chemical synthetic methods.