There is a growing list of proposed diverse biomedical applications for carbon nanoparticles, such as nanotubes and fullerenes, which possess many unique physical and chemical properties. Among them, modulation of the activity of ion channels can be predicted based on computational molecular modeling and docking [1, 2], although experimental evidence remains limited [3]. We aimed to investigate the effect of water-soluble pristine C60 fullerene nanoparticles (C60NPs) on muscarinic receptor cation current (mICAT), which underlies cholinergic excitation of visceral smooth muscles and which is mainly mediated by TRPC4 channels [4]. Experiments were performed on single collagenase-dispersed smooth muscle cells freshly isolated from the longitudinal layer of the mouse ileum, using patch-clamp techniques. mICAT was isolated using symmetrical Cs+ containing (125 mM) solutions with [Ca2+]i ‘clamped’ at 100 nM (10 mM BAPTA/4.6 mM CaCl2 mixture). The current was induced by intracellular infusion of 0.2 mM GTPγS, which activates G-proteins directly, i.e. by bypassing the muscarinic receptors. Under these conditions, mICAT slowly reached a peak amplitude of 451±52 pA (n=9) 5-10 min after break-through. C60NPs applied at 10-6 M at the steady-state response to G-protein activation caused mIcat inhibition by 47.0±3.5% (n=9). The current inhibition developed slowly, with the time constant of 119±16 s. C60NPs inhibited mICAT irreversibly and in a voltage-independent manner, as examined by applying slow voltage ramps from 80 to -120 mV. mICAT shows prominent voltage dependent properties, while its voltage dependence is additionally regulated by G-proteins [5]. Interestingly, voltage-dependent relaxations of mICAT (deactivation during voltage steps from -40 to -120 mV and reactivation by stepping back to -40 mV) became about 5-fold faster in the presence of C60NPs, an effect opposite to that seen during increasing G-protein activation by GTPγS. Finally, specificity of the inhibitory action of C60NPs on mICAT was tested by examining voltage-gated K+ currents. K+ current density in murine intestinal myocytes was 115±9 and 120±7 pA/pF (n=4; P=0.676) in control and in the presence of C60NPs, respectively. We conclude, that C60NPs specifically inhibit mICAT, but not voltage-activated K+ currents. However, C60NPs are unlikely to cause any direct block of the TRPC4 channel; rather, they may accumulate in the membrane and disrupt G-protein signalling leading to mICAT generation. Thus, C60NPs may represent a novel class of bioaccessible and biocompatible molecules for the treatment of disorders of gastrointestinal motility.