Grapevine (Vitis vinifera L.) is considered one of the most economically important crops worldwide. Vitis gene bank are traditionally kept in the field collections, however such maintenance of plant genetic resources is insecure because they are exposed to pests, diseases and other hazards related to adverse biotic and abiotic factors. Cryopreservation is a method for safe and long-term cost-effective preservation of the grapevine. Establishing a gene bank using cryopreservation is extremely important for preserving genetic diversity. The use of in vitro culture for vegetative propagation, offers an important alternative to conventional methods of plant propagation. The use of specific antioxidants in appropriate concentrations can significantly reduce the damage from reactive oxygen species, and thus improve the efficiency of cryopreservation. The aim of this study was to determine the influence of antioxidants in the microculture preculture on the growth and regeneration of shoot tips after cryopreservation. The grape varieties used in the research are: 'Graševina', 'Plavac mali crni' and 'Pošip bijeli'. In the cultivar 'Graševina', a higher percentage of plant regeneration was found on the medium with ascorbic acid compared to the plants on the medium with salicylic acid. In the cultivars 'Plavac mali crni' and 'Pošip bijeli', the highest percentage of regenerated plants developed on the medium with the addition of glutathione, and the lowest on the medium with the addition of salicylic acid. On the cultivar 'Graševina', plant regeneration was 11,1 %. The percentage of proliferated after freezing plants (PVS2 50') performed on different genotypes of ‘Plavac mali crni’ and ‘Pošip bijeli’ was between 25,0 % and 50,0 %.