The synthesis of acetoin in YM medium and under aerobic conditions by Hanseniaspora guilliermondii was studied. Experimental design and surface response analysis were employed to evaluate the influence of glucose concentration, temperature and pH on the process. The experiments were carried out in shaking flasks under controlled temperature. Five different levels of initial concentration of glucose, temperature and pH were used with variations from 17 to 81 g/L, from 24 to 36ºC and from 4.0 to 6.0, respectively. The maximum concentration of acetoin was the response obtained. The parameters which affected acetoin production most were initial concentration of glucose and temperature. An empirical model that showed statistically significant and predictive capacity was constructed. The optimum values of glucose concentration and temperature obtained for acetoin production were 63 g/L and 28ºC, respectively.