BACKGROUND: Black currants are berries that attract attention due to the beneficial effects they possess. They are proven to be good antioxidants, anticancerogenic, cardio- and neuroprotective agents. OBJECTIVE: The main objective of this research was to evaluate the differences in the flavonols composition, antioxidant and spasmolytic activity of juices obtained from four different black currant cultivars – Triton, Tenah, Ben Sarek, and Ometa, grown over a 4-year long period on the territory of Serbia. METHODS: The qualitative and quantitative determination of black currants juices flavonols was performed using the HPLC. The antioxidative activity was carried out in DPPH and β-carotene systems. The study examined the impact of the main black currants flavonols and black currants juices on the spontaneous, KCl, and acetylcholine-induced ileum smooth muscle contractions. RESULTS: The dominant flavonol in juices of the four different black currant cultivars was myricetin, with its highest content obtained from the 2015 Triton juice (820.1±34.7 μg g–1). Quercetin was the second in abundance and the lowest yields were observed in kaempferol. CONCLUSION: The investigated black currant cultivars were proven to be good sources of flavonols and potential antioxidants with the significant spasmolytic activity that could be successfully used in treating gastrointestinal disorders.