Glycerol production by osmophilic yeasts has a significant potential for large-scale production. The effects of important parameters including initial glucose concentration, carbon/nitrogen (C/N) ratio, aeration rate and pH were investigated in a 61 bioreactor with the objective of a further scale-up of the process. The kinetics of glycerol biosynthesis were analysed as a function of these parameters. The initial glucose concentration significantly affected both cell growth and glycerol accumulation. A high C/N ratio resulted in poor cell growth, slow glucose uptake and a small amount of glycerol accumulation, whereas a decrease in C/N ratio favoured increased rates of growth, glucose uptake and production of high concentrations of ethanol but not necessarily increased glycerol production. Control of pH was not necessary, but aeration rate played an important role in the yield and productivity of glycerol.