Tibicos or „water kefir“ grains consist of a mixed culture of bacteria and yeast and polysaccharides produced by these microorganisms. The symbiotic relationship of microorganisms enables balanced fermentation and obtaining the final product – „water kefir“. The grains used for cultivation was obtained from private home cultivation and their exact microbiological composition is unknown. In this paper, the optimization of cultivation conditions (temperature, different sucrose concentration) was performed. The cultivation was carried out in previously sterilized aqueous solutions of sucrose of different concentrations (20 - 100 g Lˉ¹) and at different growing temperatures (20 - 40°C). The influence of alternative substrates of sugar alcohols (erythritol and xylitol) and stevia on the metabolic activity of water kefir grains was also determined. Alternative substrates are not metabolized which makes them useful as added sweeteners in the final product formulation. Between the tested values for the fermentation temperature and different sucrose concentrations, a temperature of 30°C and a sucrose concentration of 40 g Lˉ¹ was determined as optimal conditions. The dynamics of „water kefir grain“ growth and product production under optimal conditions was characterized by some basic biokinetic parameters (YEtOH/S = 0.012 g gˉ¹, PrEtOH = 0.016 g Lˉ¹ dayˉ¹; YAceti acid./S = 0.013 g gˉ¹, PrAcetic acid. = 0.018 g Lˉ¹ dayˉ¹; YLactic acid./S = 0.028 g gˉ¹, PrLactic acid. = 0.038 g Lˉ¹ dayˉ¹).