The large-conductance Ca2+- and voltage-activated K+ channels (BKCa, MaxiK) are widely expressed in different cell types, where they provide an important negative feedback control of membrane potential by generating outward hyperpolarizing current in response to increment in free intracellular Ca2+ concentration. Correspondingly, maxi-K channels play many important physiological roles and they are considered to be important drug targets [1]. In particular, activation of these channels in vascular smooth muscles leads to membrane hyperpolarization and vasorelaxation, which makes them a key element of the regulation of vascular tone. On the other hand, carbon nanoparticles (CNPs), such as nanotubes and fullerenes, display multiple unique physical and chemical properties and hence there is a number of proposed diverse biomedical applications for CNPs, including modulation of the activity of various ion channels. We thus aimed to investigate the effects of water-soluble pristine C60 fullerenes on MaxiK channels expressed in rat pulmonary artery and mouse ileal myocytes. The structure of C60 fullerene particles in aqueous solution was studied using scanning tunnelling microscopy. Molecular modelling and docking was done based on 3NAF and 5TJ6 structures from the PDB database using SWISS MODEL, SDOCK+ embedded in the QXP package, GROMACS 4.6, and MembraneBuilder. Rat pulmonary artery rings were used in tensiometry studies, while single freshly dispersed rat pulmonary artery smooth muscle cells and mouse ileal myocytes were used for patch-clamp recordings in standard whole-cell and cell-attached configurations. Values are means±S.E.M., with differences evaluated using Student’s t-test. C60 fullerenes applied at 7.5 μg/ml inhibited the net outward potassium currents reducing current density from 132.4±9.6 to 75.0±9.0 pA/pF (n=9) at 120 mV (P=0.0005). The inhibitory effect was lost with high EGTA concentration (10 mM) in the pipette solution used to abolish MaxiK activity, indicating that C60 fullerenes specifically inhibit MaxiK, but not voltage-dependent K+ (Kv) channels. In functional tests, C60 fullerenes enhanced phenylephrine-induced contraction of pulmonary artery rings by about 25% and reduced endothelium-dependent acetylcholine-induced relaxation by about 40%. Both molecular docking simulations and analysis of single channel activity indicated that C60 fullerenes blocked MaxiK channel pore in its open state. Notably, the inhibitory effect of C60 fullerenes on the whole-cell current was due to significant reduction in channel open probability, as MaxiK single channel conductance remained unaltered: 125.2±5.5 pS in control and 125.7±9.6 pS with C60 fullerenes (n=3). Thus, we characterised new pharmacological properties and novel molecular target for this class of bioaccessible and biocompatible nanostructured materials.