BACKGROUND/AIMS: The primary cilium is a nanoscale membrane protrusion believed to act as a mechano-chemical sensor in a range of different cell types. Disruptions in its structure and signalling have been linked to a number of medical conditions, referred to as ciliopathies, but remain poorly understood due to lack of techniques capable of investigating signal transduction in cilia at nanoscale. Here we set out to use latest advances in nanopipette technology to address the question of ion channel distribution along the structure of primary cilium. METHODS: We used glass nanopipettes and Scanning Ion Conductance Microscopy (SICM) to image 3D topography of intact primary cilia in inner medullary collecting duct (IMCD) cells with nanoscale resolution. The high-resolution topographical images were then used to navigate the nanopipette along the structure of each cilium and perform spatially resolved single-channel recordings under precisely controlled mechanical and chemical stimulation. RESULTS: We have successfully obtained first single-channel recordings at specific locations of intact primary cilia. Our experiments revealed significant differences between the populations of channels present at the ciliary base, tip and within extra-ciliary regions in terms of mean conductance and sensitivity to membrane displacement as small as 100 nm. Ion channels at the base of cilium, where mechanical strain is expected to be the highest, appeared particularly sensitive to the mechanical displacement. CONCLUSION: Our results suggest the distribution of ion channels in the membrane of primary cilia is non-homogeneous. The relationship between the location and function of ciliary ion channels could be key to understanding signal transduction in primary cilia.