Biodiesel is a mixture of esters of fatty acids (most often palmitic, stearic and oleic) and lower alcohols (in our work butanol) produced by transesterification. It is a renewable source of energy, prepared from triacylglycerides, which are contained in vegetable oils and animal fats. This work focuses on alkaline catalyzed transesterification of rapeseed oil with butanol and comparison of two catalysts (potassium hydroxide and potassium tert-butoxide). In industry is usually transesterification of rapeseed oil carried out like reaction catalyzed by potassium hydroxide. Potassium hydroxide have high content of K2CO3, KHCO3 and water. Moreover water is formed by neutralization of potassium hydroxide with free fatty acids contained in oil. In cause of tert-butoxide catalyzed reaction, it is not possible because tert-butoxide have not a OH- aniont, which is needed for water forming. The influence of various conditions (addition of water, temperature of separation, intensity of stirring and type of catalyst) on butanolysis process was studied for both catalysts. For both catalysts dependence of conversions on time were plotted. When tert-butoxide was used, satisfactory phase separation was not achieved. The only way was separation of hot crude reaction mixture without adding water. Ester formed by this method had high content of free glycerol and soaps, but reached higher conversion. The best results were obtained with KOH and subsequent separation of cold crude reaction mixture with the addition of water and slow stirring. The difference between reactions catalyzed by potassium hydroxide and potassium tert-butoxide was described.