Acetylcholine and carbachol are known to modulate cell functions by binding to nicotinic and muscarinic acetylcholine receptors, located on the plasma membrane of target cells. However, several acetylcholinereceptor agonists and antagonists have been reported to modulate nuclear membrane channels as well, specifically, LCC-channels (Large Conductance Cation channels), which are one of the most common yet least studied ion channels of the nuclear membrane. In this study, we aimed to test whether acetylcholine and carbachol can interact with these channels and alter their electrophysiological properties. Rat brain cerebella were extracted in an ice-cold environment, fragmented, and roughly homogenized. Later, the homogenized samples were centrifuged, the nuclei-containing fraction was resuspended, and LCC-channels of individual nuclei were examined using a nucleus-attached configuration and voltage-clamp mode of the patch-clamp technique. It was found that acetylcholine, when applied to the intranuclear side, decreases the amplitude of currents passing through LCC-channels and the probability of the channels being in an open state (Po) at negative membrane potentials. In contrast, when applied to the perinuclear side, acetylcholine increases their Po at negative potentials while decreasing the amplitude of currents across all the potentials. Carbachol, when applied to the intranuclear side of the membrane, reduces Po of LCCchannels at -40 mV without affecting current amplitude. In contrast, when interacting with the perinuclear side of the channels, it decreases the amplitude of the currents at all applied potentials without affecting Po. These findings demonstrate that acetylcholine and carbachol have asymmetric and side-specific effects on LCC-channels at both positive and negative membrane potentials.